Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

The present invention is in the field of kinase proteins that are related to the serine/threonine kinase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Protein Kinases

Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.

The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.

The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.).

The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887).

Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a “kinase cascade”.

Another ligand-activated protein kinase is 5′-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.

The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).

PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.

The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.

Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.

Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.

Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.

LIM Domain Kinases

The novel human protein, and encoding gene, provided by the present invention is related to the family of serine/threonine kinases in general, particularly LIM domain kinases (LIMK), and shows the highest degree of similarity to LIMK2, and the LIMK2b isoforn (Genbank gi8051618) in particular (see the amino acid sequence alignment of the protein of the present invention against LIMK2b provided in FIG. 2). LIMK proteins generally have serine/threonine kinase activity. The protein of the present invention may be a novel alternative splice form of the art-known protein provided in Genbank gi805161 ; however, the structure of the gene provided by the present invention is different from the art-known gene of gi8051618 and the first exon of the gene of the present invention is novel, suggesting a novel gene rather than an alternative splice form. Furthermore, the protein of the present invention lacks an LIM domain relative to gi8051618. The protein of the present invention does contain the kinase catalytic domain.

Approximately 40 LIM proteins, named for the LIM domains they contain, are known to exist in eukaryotes. LIM domains are conserved, cystein-rich structures that contain 2 zinc fingers that are thought to modulate protein-protein interactions. LIMK1 and LIMK2 are members of a LIM subfamily characterized by 2 N-terminal LIM domains and a C-terminal protein kinase domain. LIMK1 and LIMK2 mRNA expression varies greatly between different tissues. The protein kinase domains of LIMK1 and LIMK2 contain a unique sequence motif comprising Asp-Leu-Asn-Ser-His-Asn in subdomain VIB and a strongly basic insert between subdomains VII and VIII (Okano et al., J. Biol. Chem. 270 (52), 31321-31330 (1995)). The protein kinase domain present in LIMKs is significantly different than other kinase domains, sharing about 32% identity.

LIMK is activated by ROCK (a downstream effector of Rho) via phosphorylation. LIMK then phosphorylates cofilin, which inhibits its actin-depolymerizing activity, thereby leading to Rho-induced reorganization of the actin cytoskeleton (Maekawa et al., Science 285: 895-898, 1999).

The LIMK2a and LIMK2b alternative transcript forms are differentially expressed in a tissue-specific manner and are generated by variation in transcriptional initiation utilizing alternative promoters. LIMK2a contains 2 LIM domains, a PDZ domain (a domain that functions in protein-protein interactions targeting the protein to the submembranous compartment), and a kinase domain; whereas LIMK2b just has 1.5 LIM domains. Alteration of LIMK2a and LIMK2b regulation has been observed in some cancer cell lines (Osada et al., Biochem. Biophys. Res. Commun. 229: 582-589, 1996).

For a further review of LIMK proteins, see Nomoto et at, Gene 236 (2), 259-271 (1999).

Kinase proteins, particularly members of the serine/threonine kinase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the serine/threonine kinase subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the serine/threonine kinase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the kinase protein of the present invention. (SEQ ID NO:1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland.

FIG. 2 provides the predicted amino acid sequence of the kinase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding the kinase protein of the present invention. (SEQ ID NO:3) In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 42 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a kinase protein or part of a kinase protein and are related to the serine/threonine kinase subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human kinase peptides and proteins that are related to the serine/threonine kinase subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these kinase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the kinase of the present invention.

In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known kinase proteins of the serine/threonine kinase subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known serine/threonine kinase family or subfamily of kinase proteins.

Specific Embodiments Peptide Molecules

The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the kinase family of proteins and are related to the serine/threonine kinase subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the kinase peptides of the present invention, kinase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the kinase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the kinase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

The isolated kinase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. For example, a nucleic acid molecule encoding the kinase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genornic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the kinase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

The kinase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a kinase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the kinase peptide. “Operatively linked” indicates that the kinase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the kinase peptide.

In some uses, the fusion protein does not affect the activity of the kinase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant kinase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the kinase peptide.

As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the kinase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the kinase peptides of the present invention as well as being encoded by the same genetic locus as the kinase peptide provided herein. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 22 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

Allelic variants of a kinase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by the same genetic locus as the kinase peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 22 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 42 different nucleotide positions. Some of these SNPs, which are located outside the ORF and in introns, may affect gene transcription.

Paralogs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

Orthologs of a kinase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the kinase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a kinase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

Non-naturally occurring variants of the kinase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the kinase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a kinase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant kinase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as kinase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

The present invention further provides fragments of the kinase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a kinase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the kinase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the kinase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in kinase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Accordingly, the kinase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature kinase peptide is fused with another compound, such as a compound to increase the half-life of the kinase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature kinase peptide, such as a leader or secretory sequence or a sequence for purification of the mature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a kinase-effector protein interaction or kinase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, kinases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of kinase proteins, particularly members of the serine/threonine kinase subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to kinases that are related to members of the serine/threonine kinase subfamily. Such assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase-related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

The proteins of the present invention are also usefull in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the kinase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the kinase protein.

The polypeptides can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the kinase. Both the kinases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the kinase. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.

Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway that the kinase protein normally interacts (for example, another kinase). Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant kinases or appropriate fragments containing mutations that affect kinase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) kinase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the kinase can be assayed. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.

The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase. Thus, the soluble polypeptide that competes with the target kinase region is designed to contain peptide sequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable to immobilize either the kinase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of kinase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a kinase-binding protein and a candidate compound are incubated in the kinase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the kinase protein target molecule, or which are reactive with kinase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

Agents that modulate one of the kinases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. These methods of treatment include the steps of administering a modulator of kinase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

In yet another aspect of the invention, the kinase proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693/1696; and Brent WO94110300), to identify other proteins, which bind to or interact with the kinase and are involved in kinase activity. Such kinase-binding proteins are also likely to be involved in the propagation of signals by the kinase proteins or kinase targets as, for example, downstream elements of a kinase-mediated signaling pathway. Alternatively, such kinase-binding proteins are likely to be kinase inhibitors.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a kinase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a kinase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the kinase protein.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specific antibody, or a kinase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

The kinase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. The method involves contacting a biological sample with a compound capable of interacting with the kinase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the kinase protein in which one or more of the kinase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and kinase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. Accordingly, methods for treatment include the use of the kinase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or kinase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the kinase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nuleic acid arrays and similar methods have been developed for antibody arrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid molecules that encode a kinase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the kinase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the kinase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the kinase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 22 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 42 different nucleotide positions. Some of these SNPs, which are located outside the ORF and in introns, may affect gene transcription.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to low stringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 42 different nucleotide positions.

The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 22 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in kinase protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a kinase protein, such as by measuring a level of a kinase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a kinase gene has been mutated. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain.

Nucleic acid expression assays are useful for drug screening to identify compounds that modulate kinase nucleic acid expression.

The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the kinase gene, particularly biological and pathological processes that are mediated by the kinase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland. The method typically includes assaying the ability of the compound to modulate the expression of the kinase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired kinase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the kinase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

The assay for kinase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the kinase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of kinase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of kinase mRNA in the presence of the candidate compound is compared to the level of expression of kinase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate kinase nucleic acid expression in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

Alternatively, a modulator for kinase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the kinase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant and fetal brain, and thyroid gland.

The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the kinase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in kinase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in kinase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the kinase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the kinase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a kinase protein.

Individuals carrying mutations in the kinase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 42 different nucleotide positions. Some of these SNPs, which are located outside the ORF and in introns, may affect gene transcription. The gene encoding the novel kinase protein of the present invention is located on a genome component that has been mapped to human chromosome 22 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

Alternatively, mutations in a kinase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant kinase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers etal., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 21 7:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the kinase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 42 different nucleotide positions. Some of these SNPs, which are located outside the ORF and in introns, may affect gene transcription.

Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs to control kinase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of kinase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into kinase protein.

Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of kinase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired kinase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the kinase protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in kinase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired kinase protein to treat the individual.

The invention also encompasses kits for detecting the presence of a kinase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that the kinase proteins of the present invention are expressed in humans in teratocarcinoma, ovary, testis, nervous tissue, bladder, infant brain, and thyroid gland, as indicated by virtual northern blot analysis. In addition, PCR-based tissue screening panels indicate expression in fetal brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting kinase nucleic acid in a biological sample; means for determining the amount of kinase nucleic acid in the sample; and means for comparing the amount of kinase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect kinase protein nRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

Using such arrays, the present invention provides methods to identify the expression of the kinase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the kinase gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the kinase protein of the present invention. SNPs were identified at 42 different nucleotide positions. Some of these SNPs, which are located outside the ORF and in introns, may affect gene transcription.

Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified kinase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permnits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as kinases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, which is typically the case with kinases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a kinase protein or peptide that can be further purified to produce desired amounts of kinase protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involving the kinase protein or kinase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native kinase protein is useful for assaying compounds that stimulate or inhibit kinase protein function.

Host cells are also useful for identifying kinase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant kinase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native kinase protein.

Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a kinase protein and identifying and evaluating modulators of kinase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the kinase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the kinase protein to particular cells.

Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, kinase protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo kinase protein function, including substrate interaction, the effect of specific mutant kinase proteins on kinase protein function and substrate interaction, and the effect of chimeric kinase proteins. It is also possible to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more kinase protein functions.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

4 1 2320 DNA Human 1 cccagggcgc cgtaggcggt gcatcccgtt cgcgcctggg gctgtggtct tcccgcgcct 60 gaggcggcgg cggcaggagc tgaggggagt tgtagggaac tgaggggagc tgctgtgtcc 120 cccgcctcct cctccccatt tccgcgctcc cgggaccatg tccgcgctgg cgggtgaaga 180 tgtctggagg tgtccaggct gtggggacca cattgctcca agccagatat ggtacaggac 240 tgtcaacgaa acctggcacg gctcttgctt ccggtgaaag tgatgcgcag cctggaccac 300 cccaatgtgc tcaagttcat tggtgtgctg tacaaggata agaagctgaa cctgctgaca 360 gagtacattg aggggggcac actgaaggac tttctgcgca gtatggatcc gttcccctgg 420 cagcagaagg tcaggtttgc caaaggaatc gcctccggaa tggacaagac tgtggtggtg 480 gcagactttg ggctgtcacg gctcatagtg gaagagagga aaagggcccc catggagaag 540 gccaccacca agaaacgcac cttgcgcaag aacgaccgca agaagcgcta cacggtggtg 600 ggaaacccct actggatggc ccctgagatg ctgaacggaa agagctatga tgagacggtg 660 gatatcttct cctttgggat cgttctctgt gagatcattg ggcaggtgta tgcagatcct 720 gactgccttc cccgaacact ggactttggc ctcaacgtga agcttttctg ggagaagttt 780 gttcccacag attgtccccc ggccttcttc ccgctggccg ccatctgctg cagactggag 840 cctgagagca gaccagcatt ctcgaaattg gaggactcct ttgaggccct ctccctgtac 900 ctgggggagc tgggcatccc gctgcctgca gagctggagg agttggacca cactgtgagc 960 atgcagtacg gcctgacccg ggactcacct ccctagccct ggcccagccc cctgcagggg 1020 ggtgttctac agccagcatt gcccctctgt gccccattcc tgctgtgagc agggccgtcc 1080 gggcttcctg tggattggcg gaatgtttag aagcagaaca aaccattcct attacctccc 1140 caggaggcaa gtgggcgcag caccagggaa atgtatctcc acaggttctg gggcctagtt 1200 actgtctgta aatccaatac ttgcctgaaa gctgtgaaga agaaaaaaac ccctggcctt 1260 tgggccagga ggaatctgtt actcgaatcc acccaggaac tccctggcag tggattgtgg 1320 gaggctcttg cttacactaa tcagcgtgac ctggacctgc tgggcaggat cccagggtga 1380 acctgcctgt gaactctgaa gtcactagtc cagctgggtg caggaggact tcaagtgtgt 1440 ggacgaaaga aagactgatg gctcaaaggg tgtgaaaaag tcagtgatgc tccccctttc 1500 tactccagat cctgtccttc ctggagcaag gttgagggag taggttttga agagtccctt 1560 aatatgtggt ggaacaggcc aggagttaga gaaagggctg gcttctgttt acctgctcac 1620 tggctctagc cagcccaggg accacatcaa tgtgagagga agcctccacc tcatgttttc 1680 aaacttaata ctggagactg gctgagaact tacggacaac atcctttctg tctgaaacaa 1740 acagtcacaa gcacaggaag aggctggggg actagaaaga ggccctgccc tctagaaagc 1800 tcagatcttg gcttctgtta ctcatactcg ggtgggctcc ttagtcagat gcctaaaaca 1860 ttttgcctaa agctcgatgg gttctggagg acagtgtggc ttgtcacagg cctagagtct 1920 gagggagggg agtgggagtc tcagcaatct cttggtcttg gcttcatggc aaccactgct 1980 cacccttcaa catgcctggt ttaggcagca gcttgggctg ggaagaggtg gtggcagagt 2040 ctcaaagctg agatgctgag agagatagct ccctgagctg ggccatctga cttctacctc 2100 ccatgtttgc tctcccaact cattagctcc tgggcagcat cctcctgagc cacatgtgca 2160 ggtactggaa aacctccatc ttggctccca gagctctagg aactcttcat cacaactaga 2220 tttgcctctt ctaagtgtct atgagcttgc accatattta ataaattggg aatgggtttg 2280 gggtattaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2320 2 255 PRT Human 2 Met Val Gln Asp Cys Gln Arg Asn Leu Ala Arg Leu Leu Leu Pro Val 1 5 10 15 Lys Val Met Arg Ser Leu Asp His Pro Asn Val Leu Lys Phe Ile Gly 20 25 30 Val Leu Tyr Lys Asp Lys Lys Leu Asn Leu Leu Thr Glu Tyr Ile Glu 35 40 45 Gly Gly Thr Leu Lys Asp Phe Leu Arg Ser Met Asp Pro Phe Pro Trp 50 55 60 Gln Gln Lys Val Arg Phe Ala Lys Gly Ile Ala Ser Gly Met Asp Lys 65 70 75 80 Thr Val Val Val Ala Asp Phe Gly Leu Ser Arg Leu Ile Val Glu Glu 85 90 95 Arg Lys Arg Ala Pro Met Glu Lys Ala Thr Thr Lys Lys Arg Thr Leu 100 105 110 Arg Lys Asn Asp Arg Lys Lys Arg Tyr Thr Val Val Gly Asn Pro Tyr 115 120 125 Trp Met Ala Pro Glu Met Leu Asn Gly Lys Ser Tyr Asp Glu Thr Val 130 135 140 Asp Ile Phe Ser Phe Gly Ile Val Leu Cys Glu Ile Ile Gly Gln Val 145 150 155 160 Tyr Ala Asp Pro Asp Cys Leu Pro Arg Thr Leu Asp Phe Gly Leu Asn 165 170 175 Val Lys Leu Phe Trp Glu Lys Phe Val Pro Thr Asp Cys Pro Pro Ala 180 185 190 Phe Phe Pro Leu Ala Ala Ile Cys Cys Arg Leu Glu Pro Glu Ser Arg 195 200 205 Pro Ala Phe Ser Lys Leu Glu Asp Ser Phe Glu Ala Leu Ser Leu Tyr 210 215 220 Leu Gly Glu Leu Gly Ile Pro Leu Pro Ala Glu Leu Glu Glu Leu Asp 225 230 235 240 His Thr Val Ser Met Gln Tyr Gly Leu Thr Arg Asp Ser Pro Pro 245 250 255 3 59065 DNA Human 3 tcatccttgc gcaggggcca tgctaacctt ctgtgtctca gtccaatttt aatgtatgtg 60 ctgctgaagc gagagtacca gaggtttttt tgatggcagt gacttgaact tatttaaaag 120 ataaggagga gccagtgagg gagaggggtg ctgtaaagat aactaaaagt gcacttcttc 180 taagaagtaa gatggaatgg gatccagaac aggggtgtca taccgagtag cccagccttt 240 gttccgtgga cactggggag tctaacccag agctgagata gcttgcagtg tggatgagcc 300 agctgagtac agcagatagg gaaaagaagc caaaaatctg aagtagggct ggggtgaagg 360 acagggaagg gctagagaga catttggaaa gtgaaaccag gtggatatga gaggagagag 420 tagagggtct tgatttcggg tctttcatgc ttaacccaaa gcaggtacta aagtatgtgt 480 tgattgaatg tctttgggtt tctcaagact ggagaaagca gggcaagctc tggagggtat 540 ggcaataaca agttatcttg aatatcctca tggtggaaag tcctgatcct gtttgaattt 600 tggaaataga aatcattcag agccaagaga ttgaattgtt gagtaagtgg gtggtcaggt 660 tacagactta attttgggtt aaaaagtaaa aacaagaaac aaggtgtggc tctaaaataa 720 tgagatgtgc tgggggtggg gcatggcagc tcataaactg accctgaaag ctcttacatg 780 taagagttcc aaaaatattt ccaaaacttg gaagattcat ttggatgttt gtgttcatta 840 aaatctctca ctaattcatt gtcttgtcca ctgtccgtaa cccaacctgg gattggtttg 900 agtgagtctc tcagactttc tgccttggag tttgtgagag agatggcata ctctgtgacc 960 actgtcaccc taaaaccaaa aaggcccctc ttgacaagga gtctgaggat tttagaccca 1020 ggaagaatga gtgatgggca tatatatatc ctattactga ggcatgagaa gagtggaatg 1080 ggtgggttga ggtggtgttt taaggcctct tgccagcttg tttaactctt ctctggggaa 1140 cgagggggac aactgtgtac attggctgct ccagaatgat gttgagcaat cttgaagtgc 1200 caggagctgt gctttgtcta ttcatggccc ctgtgcctgt gaaacagggt tcggtgactg 1260 tcactgtgcc tgtggcagtc tgtagttacc cagagagaac aaagctgcat acacagagcg 1320 cacaagggag tcttgtaaca accttgtcct gctttctagg gctgagtcag gtaccacagc 1380 ttgatctcag ctgtcctctt tatttcaaga agttgacatc tgagccatac caggagtatt 1440 gtattttgtt tgaggcctct ctttttggag gaacatggac cgactctgtg cttttgtcta 1500 tgctggtctc tgagctcaca caacccttca ccctcctttc tcagccagtg ataggtaagt 1560 cttccctatc ttgcaaggct cagctcaagt gtcagcttcc tctacaaaga ctttcctggt 1620 tcccctcatt ggagtgaaca agagttgaca tggtagaatg gaaagagcag aagctttaga 1680 atgagccaga cctgagtatg aatgctagat ccaccactta gctagtcaac cctgccccct 1740 gcctcaagtt ttaattttcc tatccattaa gtgaatataa taatacctgt gtcacaggat 1800 tattttgaga attaaatgag attaggtcta tgaaagcacc tagcagagtt cttggcatat 1860 aggaggcatt cattaaatat ttgttcttcc ccttttatac ccattacttt tctttttctg 1920 aactaaaata atacttggtt ctatctctga aataacatcc aagtgaaaaa tcaacaacat 1980 gaaagagcag ttcttttcca gtggatttgc ttcttaagga gcagagatta tgtaatctaa 2040 cagcctccaa catacaaaga gctttgtatc tagaacaggg gtccccagcc cctggaccgc 2100 caactggtac gggtctgtag cctgttagga accaggctgc acagcaggag gtgagcggcg 2160 ggccagtgag cattgctgcc tgagctctgc ctcctgtcag atcagtggtg gcattagatt 2220 ctcataggag tgtgaaccct attgtgaact gcacatgcaa gggatctggg ttgcatgctc 2280 cttatgagaa tctcactaat ggctgatgat ctgagttgga acagtttgat accaaaacca 2340 tccccccgcc ccccaacccc cagcctaggg tccgtggaaa aattggcccc tggtgccaaa 2400 aaggttgagg actgctgatc tagaggacca atttattcaa tgttggttga gtaaatgagc 2460 tcttggatta ggtgatggaa aaatctgaaa aaacagggct tttgaggaat aggaaaaggc 2520 agtaacatgt ttaacccaga gagaagtttc tggctgttgg ctgggaatag tcataggaag 2580 ggctgacact gaaaagaagg agattgtgtt cgtttcttct tctcagagct ataagcaaag 2640 gctgaaagtt ctagaaaaag gcaagttttg tttcagtaga aaaaaggata atcagaacca 2700 tttttagaaa atggaatgag actacttttg aggccatgag ttccttgtcc ctggagagat 2760 gagcagaggt tggacaagtg cttaccagag atcttgtgga ggcagaaact gtgcatctag 2820 cagagcattg gcctaaccct ttcaaatgag atgctgttaa ctcagtctta ttctacatgg 2880 taggaatcct gtccctttgc ctcctgctac tttgggcctc tcaacctctt ggttttgtgt 2940 gcaggtgaag atgtctggag gtgtccaggc tgtggggacc acattgctcc aagccagata 3000 tggtacagga ctgtcaacga aacctggcac ggctcttgct tccggtaggt gggcctatcc 3060 tcccatcttt accagtgtac tatgggccaa gcactatttc atgttctgat ggaaaacaca 3120 gaaacaagct tctgagttga gaatttcaat cttagggtgg ggaaaggaat gtaccaagga 3180 agagctcatg accaaacctc aagtgtggcc cccctgaacc caggttaaat tggaagagcc 3240 ataaatgggc cagctggagg cagggtgggg ggatgagagg agccctttcc agggttgtcc 3300 catatccctc actttatggg tgaggaaact gaggcccagg aagagtgact ttcctgtggc 3360 tgcactacag attatgcagg tacttcaaga gttgtttgta ttcttatttt attttatttt 3420 attttatttt attttatttt attttatgag agggattctt gctgttgccc aggctggagt 3480 gcagtggtgc aatctcggct cactgcaatc tctgcctgct gggttcaagt gatttttctg 3540 ccttagcttc ctgagtagct gagatgacag gcacctgcca ccatgcgcag ctaatttttg 3600 tattttagtg gagacggggg tttcaacatg ttggtcaggc tggtcttgaa ctcctgacct 3660 caaatgatgc acccacctcg acctcccaaa gtgctggaat tacaggcgtg aaccactgtg 3720 cccagccaag agttgttttt agtgtggttg gcagagccag ctcttccttc accacaggat 3780 gcctccctag gttcctactt tttgttacta gcttttatta tagctatatt attattatta 3840 ttattattat tattattatt attattgaga cagagtctcg ctctgtcgcc caggctggtg 3900 tacagtggtg cgatcccggg ctcactgcaa cctctgcctc ccgagttcaa gcagttctcc 3960 tgcctcagcc ccccgagtag gtgggactac aggcgcctgc caccacaccc ggctaatttt 4020 tgtattttta gtagagacgg ggtttcacct tgttgaccag gctggtctgg agctcctgac 4080 ctcaggtaag tgctagaatc acaggcgtga accactgcgc ccagccaaga gttgttttta 4140 gtgtggttgg cagagccagc tcttcctcac cacaggttgc ctccctaggt tcctactttt 4200 tgttactagc tttattatag ctacattatt attattattg ttattattat tgagacagag 4260 tctcgctctg tcgcccaggc tggtgtacag tgatgtgatc ttggctcact gcaacctctg 4320 ccccccgagt tcaagcaatt ctcctgcttc agccccccta gtaggtggga ctccaggcac 4380 ctgccaccac gcccagctaa tttttgtatt tttagtagag gcggggtttc accttgttgg 4440 ccaggctggt ctcaaactcc tgacctcagg tgatccgcct gcctcggcct cccaaaatgt 4500 tgggattaca ggcatgagcc accgcgccct gcctatagct acattatttt tgtaggcagc 4560 tcagtttctt aaaaattata cagacttcaa atcagatttg ttcctgctgt ctgaggctca 4620 gtttcttcat ctggaaaatg gatggtaata atcttgttga gattgaatga aataatatat 4680 gcagtgtatc cagtacatgg tagacaccca gtgaatggtt attccttcct cccatcggat 4740 tggaattctc aagggtggga acttgtcttt atattcttca caacgtaaaa tagttgaaat 4800 ttgttggtgg aaagaagagc agtccactcc agaggctgga tgggcatgcc tggcccccaa 4860 ggtctgaagt ggtagggctg tgcctatatc ctgagaatga gatagactag gcaggcacct 4920 tgtgctgtag attccagctc ctgcacatag ctcttgttgt aaaacatccc tgtgcttata 4980 ccaagtaatt gagttgacct ttaaacactt gcctcttccc tgggaaccat ataggggatt 5040 ggcctggaga cgtctggcct ctggaagagt tggaaagcag ccatcattat tatcctttcc 5100 tttcagctat aactcagagc tctcaagtct tttctgtgga tcttattgcc ttggttcttg 5160 ccccttttac tcccagggaa gttgattctg tcttttctgt tccatttagt atgacaggag 5220 cagagaatgt cagagctgta agggacctta tagttaaagc ctttggctgg tcctttcatt 5280 ttatagctgg gactaataag taacgtcaaa acccaatgag ttcacagatt gggtctcgcc 5340 ttggcatgta acccatatgt tcatattctt gctgttttcc tatgtgtatg aatattttct 5400 atccaaaata agcaggacag ggtagagcaa gttaatcttt ggaatttctg gattctctta 5460 gagctaaaaa acttcagaac tagaagaaac cacccactat atggtataac ccattcatat 5520 cacagatgag gcctgaaacc aaaaagactt gctcaggcca tggatgacaa gagctggccc 5580 tagcactgaa ctcttgggtc atttgtaggt ctagtcagat gctagcttgt tagctctgtg 5640 cgtgcgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgagat agagacagaa agataacata 5700 tgtacacaaa tacataaaga ggaagtagac acgttagcat ggtagataag agtacaggca 5760 ggccaggcgt ggtggctcac gcctgtaatc ccagcacttt gggaggccaa ggcaggtgga 5820 tcacctgagg tcaggaattc gagaccagcc tgaccaacat ggtgaaaccc catctctact 5880 aaatacagaa aaaaattagc ttggcatggt ggcacatgcc tgtaatccca gctacttggg 5940 aagctgaagc aggagaatcg cttgaatccg ggaagcagaa gttgcagtga gccgagattg 6000 tgccattaca gtctagcctg ggcaacaaga gggaaactcc atcgcaaaaa aacaaccacc 6060 accaagagta caggctatgg aatgagacta tggttttaaa tcctggcttt gcaatttatt 6120 aactagcctt aagtgacttc cctgagcttc aggcaccaat ctgtaaaatg aggataagaa 6180 tattactcat gccacatggt tgttagggag gattaaatgt gataacctat ataaagtggc 6240 tagcatagca tctgacatat agaaaactct taatagggcc ggacgtggtg gcttatgcct 6300 gtaatcctag cactctggga ggccgaggca gaaggatcgc ttgagcccat gagcccagga 6360 gtttgagacc agcctggcca acatggcaaa actccacctc tacaaaaaat acaaaaatat 6420 tagccaggcg tgatggcaca cacctgtagt cccagctact tgggaagctg aggagcgatg 6480 attacctgag cccagggata tcaaggctgt agtgagctgt gatcatgcca ctgtactcca 6540 tccagctggg ggacagagtg aaacccctgt ctcaaaacaa aacaaatgaa aaaaaaaacc 6600 cttaataatc agtaactgtc actttatatt atgttgtgag tgtgtgtcta tatacaccta 6660 tatgtataca tttctcttat tacacattca ttggtgatct gatgtggagc cccagggatt 6720 aagggcaact ttgaactacc ctgacacaat caagccaaat atcattcccg tggaggaagt 6780 agagtatcta ggttctgtct cctagttgca gctttacctt gaggacagag actctaatcc 6840 agctgtgctg aaggagcaca tctcctgact tctgagcttt cccctggtaa attcaaactg 6900 gatgtcacgg cgccctcaga tagagcctgg taatttgccc tggggagagt gactgtcttt 6960 tggatctaat ttgacttttg ccccagttgg aggaaaatct tcagggctag gaaggattgt 7020 atttgtctga ccccagagat aacctgggtt ttgaggaaca tggggcatca acctgaatgg 7080 tcttgtaaga tctctcccac gccagcttgc cagtgtttct ctgatgaatt tagagtacct 7140 gagtagtgca ggcctgctgg gaggaggact ctccctctgt gctactcaga gaaattcatt 7200 cttcaaggcc cccttccagc cttgctctta cccagctggg ctacagttac aataaaggaa 7260 atgacttttc ttctcccctt cccccagtac ctttgttttc ctagtcacag ggtggggctg 7320 gatattgaat ggagaaattg ctggggtcca tcctaaactc ctcccctcat ctctccctta 7380 cattacccca ttcttctgtc tgcagccaca tccataatcc tgcctctgtt agccttccga 7440 cagaccctca ggtgcccagg acaacaggaa gctacttaaa gctggaacct cagactgtgc 7500 aatggaggcc agtgacaaaa ctgaaagtag ctctgtcagt aattgtgctg gtgcgattag 7560 gcagctggcc agaatctttt ggatctcctg gacatatggc tgactagtcc tcccaagcct 7620 tcccaacagg cctctttttt ttcctttttt tcttttcttt tttttctttc tttctttctt 7680 tctttttttt ttttttttag gctagtgaag tgaaattgtg ggagtggaaa aggaacaaag 7740 aaatcggtaa ctggtagtga tcaattactt gtaaacacta ttgtacttgg accagcccag 7800 taggcctttt ttaaaactct gagttacctc tctttccttt ccttgagcag tgccattaat 7860 tctgtatctg gggcaatcct ttctgatgtt ctctggacct ggctctctct ccttaggaga 7920 ggccaggaga gtagccagag agcatgtcat ttgtagctga ggttaaagtg tggagctatc 7980 aatggtgacc tggcctcttg gcatgttagc aagccagagg accttgacaa cttttttgat 8040 gattgtccgt tcaccctgat caaaggtgtt tggcttagga ggagggaaga aaagctaccc 8100 ctattagtct tgatggcccc agcgtgggtc tctattgctt gacctggttc ctagcagcat 8160 tatcagaagg aaaatccacc gctcttaagg ctcctgggaa ctttcaggac ttcctttctc 8220 aggattgcaa acataagact atttgagctt tcacttttga aaagcggtta ctaataccta 8280 tactctggga aagggctaat gcagatagaa gactgtggtc actgcatcag gcaacagacc 8340 atttccgcta aatttagtga ctccaggaag gccagtgaag aaataacaca cgtagcaacc 8400 agagactgtg ttgtaatatg ttggctgaca gcagggtact ttctgtgatg ctgaaagcca 8460 cattcatttt ctctcccctc atccccatct aagcaagcct ggtagaatca taattacagt 8520 aataggtacc acttattgag tactctgtgc cagacaccct cctgagcata cgacatgcat 8580 agcacattta atccttacaa tgacttaata aaatgtagta ctagtcttac ctacttcgag 8640 aatagggaaa tggaggttac ttgtttaaag tcacagagct aataggtagc atagctgaga 8700 tttgaactca ggcattctta ctccttgcct gcaagagtct cttggcattc ttgaatgcaa 8760 gcatatttct taacctcact gaggctcagt ttcctcttat ataatatggg gtaaagagcc 8820 ctcaccctgc ctgccacaca ctggtagtgt cagataacat tgaagggtgt tagtttaaag 8880 gcttcatgga ctctataatg tcaacaaaag tgctgttaac tttcttctgg gtctcaggct 8940 cctgatgtag agtcagtgga gcaaccctgc catctgctgt tatgctgttg atgttgctgc 9000 cacacttact aacctaaacc tttgattctg gctgtggcct tctccagaag gtgtttactc 9060 atttgtccag tttatctttt aggaaacagc cagcccgtag atcattaagg ctggctattg 9120 gacagggggc tggggcctgc ctgacagagg aaggaagggc agacatctgg ttcttcctct 9180 gcccctacaa gagactccag cctgaccaca gagtggtact cctaggatgt agcagcagca 9240 tatgagcttg aatgtgcctt aatcctgctc tttactttga gaagagagaa ctaaggaccc 9300 acagatgttt cacagcttct ataggaggca gaggtagaaa aatggagaga gatgaggcca 9360 gagatagata actgatatta attaaacgtt gtattaagaa cctcacttag attatctgat 9420 tcaatcttca taataaccct gcaaccccca cctttttttg agaacagggt cttgctctgt 9480 tgtccaggct acagtgcact ggtacaatca tagttcactg cagtgtcaac ctcctgagct 9540 caagcaatcc tcccacctca gccttgcaag cagcttggac tacaggcgtg ccaccacacc 9600 ttgccatttt tttttatttt aagtagaaac aaggtcttat taatactatg ttgcccaggc 9660 tggtcttgaa ctccagcgat cctcctgccc cagcctccca aagtgcttgg gattacggaa 9720 gtaagccact gtgcctggcc agtgcaaccc ccattttata ctaaaacagg aaggcccaga 9780 aaggtttgga gtaacttgtc cagggtcaca cagatgatat ttgaactcag gtctccctgg 9840 ctcccaagag agtctgcttt ccactaggac tcccaggaga aaaaaaaaaa aaaaaacagt 9900 agacttggag acagaaaatc tgatttgagt cttagttgag ctaggctaac tgtgtaactg 9960 tgggcaagtt ccttagcccc tgtgagcctc agtttcttat ctgtaaaatg tcataaaaga 10020 aatccatctc atggagtagt tgtgatgatc aaggactctg aaaacattag aatggtttaa 10080 tgtgaaggat tagcagcagc acatggcaac attgtgcatc ttatattaac tatccaaata 10140 tatcaagcgt catttgctat atataaaagt catcaaatta ggcactgtgg gggatacgga 10200 gttggcatac tagcctggcc tcttaattaa ttcattaatt agcttattta tttttgagat 10260 aggtcttgct ctattgccca ggctggagtg cagtggcatg atgatagctt actatagcct 10320 caatctccca ggcttaaaca atcctcctga gtagctggga ctacaggcac acactaccat 10380 gcccagctaa ttttttttta attttttgta gagacagggt cttgctctgt tgcccaggct 10440 ggtctcaaac tcctgggctc gagatcctcc cacctgggcc tcacaaagtg ttgggattac 10500 aggtatgagc cacggcacct ggcctggtct cttaactggt tccctaagac agctggaaat 10560 agagaatgtc atggagcatt cctaaccatg ggctccagcc tggctttcat tctgtttctc 10620 ccctgaaaca acattccttt agtaatattc cgaataacag cttcatcagt ctgtctaccg 10680 accactcttc aggcttcatc ttatatgacc tcccaaactg cactaagggt tgtattagag 10740 aaaagtggat aaagttcgga gtcaggctgc ttgagcttaa atgccagctt cacttaccag 10800 ccacctgacc atgagtcagc tgcttaacca ttctttgcca cagtttcctt gtctatgaaa 10860 agggaaatgg ctcccacctc aaaaagttgt taacattaaa ttcaatcatg tattcaaagt 10920 cctgagcaga atgtctggcc atgactggga cttaacagat gttagcattt attattagta 10980 tctgtcagtc ttgaaatgtt ctcttccctt ggctttcatg acattccaca ctctcctggt 11040 tttctcttac ctctctggta atacctgttt gcttatcctt ctttgtccag ctctgggatg 11100 ttaccattcc ttcaggcgtg ctgttttctc cttaggcagt cttacacaca ctcatgactt 11160 ccttccattg tcctccacac actgatgacc ctaaaatcag tatctccagc ctaaaccttt 11220 ccactgagtt ctagacccat atgttgtact atcaacctgg cttgtccatt tgaatgtctt 11280 ccaggcactt cagactctct tctctagact ttgctggact ttcactcttc cccctaaaac 11340 tggctcctct tccactgaaa catgtatgtc attgagaggc accaccatcc acccagtgcc 11400 taagccagaa acctaggaat ccttgatacc tgttctctct catcctgcat atccaagcct 11460 atcagtttta tctctaaatt atattttggt aggtttactt ctttcctttt ctcccaccac 11520 caccctgctc caagctacca tcatctcacc tggatgtctg caatagcctc atctcccaca 11580 gccactctgc accccctaat ctgttctcta tagagcagtt ggaaggagtg atttttgttg 11640 tttgttttgt tttgttttag acagagtctc actctgttcc ccaaggctgg agtgcagtgg 11700 cacaatttcg gctcactgca acttctgcct cccgggttta agcaattctc ctgcctcagc 11760 ctcccaagta gctgggatta aggcaccggc ccccataccc agctaatttt tatattttta 11820 gtagagatgg ggttttgcca tgttggccaa gctagtctcg aactcctgac ctcaagtgat 11880 ccacctgcct cggcctccca aagtgctggg attacaggtg tgagccactg cacctggctg 11940 gaaggagtga tcttaaaaaa aaaaaaaaca aaaaaaaact tgactgtgtc actctgtgtt 12000 gtctctccta ccttgtatac ttccacaact tcccagtgtt cttggataaa gaccaaaatc 12060 cttaacttgg ccaggcgcgg tggctcacac ctatcatctc agcactttgg gaggccgagg 12120 caggcagatc atgaagtcaa gagattgaga ccatcctggc caacatggtg aaaccccatc 12180 tctactaaaa atacaaaaat tagctggtcg tggtggcgtg tgcctgtagt cccagctact 12240 tgggaggctg aggcaggaga atcacttgaa cctgggaggc agaggttgca gtgagcccag 12300 atcacgccac tgcactccag cctggtgaca gagtaagact ccatctcaaa aaaaaaaaaa 12360 aaaaaaaaaa ttccttaatt tggcctacag tagagccctc cgtaatgtgg cctctctcca 12420 catctccaca acctcctgct ccctgcactt cagcctcacc tctcttctgg acaggccctc 12480 cttctgacaa gggctttgtt cattctgctc cctctgccta gaatgccccc ttactctgtt 12540 cacttaactc ctgcttatcg tttagatctt tacctggatg gctcagagaa atatagaagt 12600 aattcctcac cctgaaaaat aggttaggtc cctgttttat gttttcatag acctttcctt 12660 tgaggctttt tttaaaaaag tagttttaat ctcacattta ttcatgtgat catctcctta 12720 atgatatctt aagacctcta atagaacaat ttggtcatgg actgtggggt ttttgcccct 12780 cattgtgtca gcactgagca tattgttggc ataggaggga tatttgttga atgaattgct 12840 agaggtggcc aagagatatg atgtaagtca ggcttttccc tgcccttccc cttccccttc 12900 cccacatcct tcctatagca gccaccgtgg ctgcagttac tgtaaatggc aagacggaat 12960 cagttccgga cattgggttg ttttagaaaa ttgcctgcaa gtgtcagggt gataagttaa 13020 agctttgtct tttgccctca gaggagctat cccatagtga gtagaagcca gagaagctga 13080 ccccaggagt ccttctttcc agcagcaggt cttgagctgc acttctctgt agctacaatc 13140 caggcaggaa caagccctag gtacctccgg agaggagggc aagagaggaa gaatgagttc 13200 agctactcta gccaccaaac tgattatgaa ttgccctgaa atctgaaaaa tttcaattcc 13260 aatcgtaagt ttgttttgtt tcattttgtt ttcttaaatt gtatatttga aagatggcat 13320 taactaaaga tatatattca atatagagtg gaaaaaatgg aatacttgca tagtatcttt 13380 tacttatagg tgatttatga tggggagtgg ggtggatagg ttggcagttc ccccaagaag 13440 ttggaaatga agtttgtcct ctgtgagttg aactaattag atccacaagt aatgaaagca 13500 gtattgtgtt gtagttaaga gcacactcta gaaccagatt gcttagtttc aaatcctggt 13560 tctgcctttt attatctgtg tactttgggc aagttacttg ccctttgtgt gcttcatttt 13620 tctcatctag aaaatggaga ggccaggcgt agtggctcat gcctataatc ccagcacttt 13680 gggaggccga ggcgggcaga tcacctgagg tgagaagttc aagaccagcc tggccaacat 13740 ggtgaaaccc tgtctctaca aaaatacaaa aattagccag gcatgatggc gggtgcctgt 13800 aatcccagct acccaggagc ctgaggcggg agaaacactt gaacctggaa ggcagaggtt 13860 gtagtgagcc aggattgcac cactgcactc cagcctgggt gacaagagct agactcagtc 13920 taaaaaaaaa aaaaaaaaac aaactggaga tacaggctgg gtgcagggct tacacttata 13980 atatcagcac tttgggaggc ctaggcggga ggattgcttg aactcaggag tttcaagatc 14040 agtctgggta acagagcaag acctcatccc cacaaaaaat caaaaattta gccaggcatg 14100 gtggctcatg cctgtggtcc cagctactca ggaggctgag gcgagaggat tgcttgagcc 14160 caggaggttg aggctgcagt gaaccatgac tgcaccacta catgccagcc tggatgacag 14220 agcaagaccc tatctcaaaa aaaaaaaaaa aaagaaacga gccaggcgcg tttgctcacg 14280 ccagtaatcc cagcactttg ggaggccaag gcaggtggat cacttgaggt caggagatcg 14340 agactagcct ggccaacatg gtgaaacccc atctcaactg aaaatacaaa aattagccag 14400 gcatggtggc atgctcctgt agtcccagct actcacttgg aggctgaggc acgagaatcg 14460 cttgaaccca ggaggcggag gttgcagtgg gccaacatca tgtcactgca ctccagcctg 14520 ggagacagag cgagactctg tctcaataaa taaataaaca taaaataaaa taaaataaaa 14580 taaaataaaa taaaaaaata tggaggccag caggcacggt ggctcacgca tgtaatccca 14640 gcactttggg aggccgaggg gggcggatca caaggtcagg agatcgagac catcctggct 14700 aacacagtga aaccgcgtct ctactaaaaa tacacaaaat tagccaggca tggtggcagg 14760 cacctgtagt ccctgctact caggaggctg aggcaggaga atggcgtgaa cccgggaggc 14820 ggagcttgca gtgagctgag atcgcgccac tgcagtccag cctgggcgac agagcaagac 14880 tctgtctcaa aaaaaaaaaa aaaaatggag gttgggcgcg gtggctcgcg cctgtaatcc 14940 cagcactttg ggaggtcgag gcgggcggat cacctgaggt caggagttcc agaccagcct 15000 ggccaacatg gtgaaacctt gtctctacta aaattacaaa aattagccag gcacgatggc 15060 aggcacctgt aatcccagct acttaggaga ctaaggcagg agaatagctt gaacctggga 15120 gatggaggtt gcagtgtgct gagatcgcgc cactgccctc cagtagagtg agattccgtc 15180 tcaaaaaaaa aaaaaaagaa gaaatggaga tacaaactta ctacctacct ccttacaacc 15240 taccctcaca gtattactgt gaataaaagt gtgtgtagca ctgggaacac tattcacaga 15300 gcactcatga atgtttgttc tttgttatta gttactagag aggcaaatgt ctgccagggc 15360 tgaataatat gtgtgaattg gtgattgtcg cacatatcta aagaagtagt tatttttttc 15420 aattaaaact tagtttaaaa accaatataa ggccgagcgc agtggctcac acctgtaatc 15480 ccagcacttt gggaggccga ggtgggcaga tcatttgagg tcaggagttc gagactagcc 15540 tggccaacat ggtgaaaccc tgtctctgct aaaaaaaaaa aaaaagtaca aaaattagcc 15600 aggcatgatg gcaggtccct gtaatcccag ctacttggga ggccgaggca ggagaattgc 15660 ttgaacccag gaggtggagg ttgtagtgag ccgagtttgt gccactgcac ttcagcctgg 15720 gtgacagagg gagacactgt ctcaaaaaaa aaaaaaaaaa accaaaacca atataataaa 15780 taagtggcca gcaatgaaac agaaagtgaa aagttagtga agcaaaacta gtactgtatt 15840 cagataaaga tgctgaatct agatttggtc accagaatag ggtcctttgt ggcaacctgg 15900 gctagtttgg ctgactcacc actgccagga tgaaatttct ttcagtggct actcatttcc 15960 ctttatttta agtccatgct cacagagcaa ccttctgatg cctaattcag cttcctggga 16020 tacttaataa caggaagggt ctggaagtag tacctgtata ggggatatga gtgttctgat 16080 tttaatagtc aattcataag tgtacagagg gtttgataaa tggttaggtc agaaccatca 16140 cagaatgtct acacctcttt ggacattagg aaggtcaaaa acctgaaagg ccaaaagcta 16200 ggcctagatt agggtcattc accaagaaaa catcagcctt gaagagttct ctgggtggtc 16260 caccagtcaa ccttcctttg atcacacctc cttcctcgtt gcttctttaa gcattgacct 16320 gtaatgggta tggaattttt tgctcaccta actccttcct tttacagagg aagaagttga 16380 agcccagaga gatttaatgg cttgcctaag atcacacgca gattttctgt taaccagggt 16440 gatttttcag gtgttccctg ccagacgagg gcttttttcc ttgaattgcc tagagatttc 16500 ttgagatatc cgaagcattt ttcccagtgc agcctggaga aggatgtccc tgtcaacaca 16560 gcatttgtta ctcaatgtta gacattcaat tttctaatta gtatcatgga gcaacagtgg 16620 atgattatct ataaggggtt gcaattccat gcttatgtgc ttacagccca tatagacaaa 16680 tatcagctgt taaaatgaca aggcagtaga gatgtggccc caggacaaag gcatactctg 16740 ctgttagtga acactagttg gccagcaaat ttcacatggg catatacacg gccaactgta 16800 gactttaggc atttataccc attcagagag ccaaactggc aactaaagat cagcattctc 16860 tttggcattt cagctttgcg ttctgttaaa aatcactgct tgcttaaata cctctgatag 16920 ctcttcactg cctgtaggca actctttagc ctagcagact tggtctttag tgctctgccc 16980 ctactctctt ccaccattct ggcctcctgt ctaattgctg cccatatgtg ccatgcacta 17040 gagcttacag acctgctcag cgttatatga gcataccata ctctttatgc ctcagtgcat 17100 ttgcacatgt tgttccttca ggccagaatg cctgttactg cctggcaatc agcctattag 17160 agtctgccaa taccatccca tcttctgtgg aggagccccc cgccaaatcc acccatacct 17220 ctccccacca atcagagact tcttctctct ttgttattct cttcgttatt ctcttcatac 17280 ctcagttata tccatttcag tatttgttta cacatctagc atcactctta gagtgtgaaa 17340 ttctccaagt gtggagccgt atctagtttg tctttgtatc ccagagctta gcaaagtgcc 17400 tagaatgtag tgggtgctca gagtgtttgc tgggtgaatg atgtatttgt tgaacgactc 17460 tttggacact tgaataaagt ccatccagta tgcaccatta ccatctcttc gctctacaat 17520 attcttttag gcaagagctt atcttttgag gtgataagat aagctcaaac ttatgtagac 17580 taagacctca gtctgtaaat gtcatcccta agtcttaaac catcaaaacc agggcctcaa 17640 ggaatggcat gccttctgca actgtagcaa cctgctgtgc ttattttgcc gtgtttttca 17700 tttttccccc aaaagctaga gtcccttctc ccatgggcag tgctggaagt gtgctaacaa 17760 attctttctc catactgctt acgattacaa aaaaaaccct cagcatctca tgccagactt 17820 gagttaaggt tgttttcttt tgtgtgtcag ctgtattctg gtcatgactt cctgatgatg 17880 ccctatagag attttgctga gatcagaggg tgctccactg ccatcagtag cactgactct 17940 tgcagaagca ccgtttctga agttggctaa tgtcatccct cacgtttgtt tgtttgaaat 18000 ttgttttagt tccagagata gcactttcat ggaatgacgc tatcttctag aatcactttt 18060 tttttttttt tgagttggag tctcgctgtg tcgccaggct ggagtgcagt ggcacaatct 18120 cagctcactg caatctccac cttccgggtt caagtgattc ccctgcctca gcctcccgag 18180 gagctgttac tacaggcgca cacccccact cctggctaat tttatgtgtt ttagtagaga 18240 cggggtttca ccgtgttggc caggatggtc tcgatctcct gactttgtga tctgcctgct 18300 tcagcctccc aaagtgctgg gattacaggt gtgagtcacc gcgcctggcc tagaatcacc 18360 tttttatacc ataacgtgag caccactgcc gcgtcaccaa ggaaagagag aggcagctac 18420 tgtggggtta caaatgggta agagtggcac caggaaggtg aaagtctcta cttagccaag 18480 gcttaacaaa atgtcaatca ccaaacattt atttattaag ctacgttcag gataagaaga 18540 tgaacaagct atctgtacat tcattttctc gtttgtaaca aggtaatgat agtgatctat 18600 cctgcctgcc tctgagggtt attgtgagaa taaaatgaaa tcaagtggaa aagcacttag 18660 gaaaaagaaa agcattggtt ttcaattgtt agtgtggatc agaaacactg gggcttgttt 18720 aaaatgcaga ttcttagccc cagtctcagc gattctgatt ctgtatatct gaagtgggac 18780 tcaggaatct tgattttcaa caagctgacc agagggtcca atgctgctat tcctttagtt 18840 acactttcag aaatattact gtaaatcaaa tggcaagaat aaaatagtta tttgaggcag 18900 ttttagtatg ttggacctgg agtccaaaga cttgggtcaa actccagctt tgtcagttcc 18960 tagacctgtg accttaaaca gcaaccttct ctgtgaacct tagttccctc aggaacggct 19020 ctggtcacct cctgctgtac tccattgatg actcaccaca taaggctccc tgggagtccc 19080 ccaaaccttt gctctcttaa ctccttttac agcctcctac atctcctgca ggtgctgtct 19140 tctcctcctt tttccaggcc ctgctctgac acagcattca ttctcctctg ggaagggttc 19200 cttcaatgtg tctccaagca catcacaccc aggaaggacc ctgtggccat atctgtctat 19260 caccagatca aactacgtga aggcaggcac taggtactgt cagtgcccag cataggcctg 19320 gcccatacca ggtgtccaca gatgcctagt aaagaaacct atgattcagg acccccatga 19380 tgagcaacta tagcactaga acagtgataa taactaatgt ttataatgca tcttcagttt 19440 acagagggct tttgtactca tcatctagtt tagttcctgc aacaacctct tgaggaatat 19500 agcacaagca ggacaaggga agcccagaga tgttaaataa tttatccaag tttatgctgc 19560 tgggaagggc agcactgaaa ttaaaagaaa agttttctga gctcaaatcc catgcccttt 19620 cctcaatgtg agctctagca aggtattcag gaatcctgcc tctacagttc agagcctcaa 19680 attgctgggt atgttgagtt cttgtatctg atttttctag atttcctgcc cacattctta 19740 ctgtctggat atcaggaaag agtttatcaa atgcctgtgg aaatccaaga taaggtctca 19800 tgatgagtaa cccagtgaaa acatgaagtc aagtctaact agtcactact atttcactac 19860 tgctgactcc tgatgatcag ctccttttct aagtgcttac tgtccactta ttccatcatc 19920 tgcctagaat ttatgtgaag gaatcaaagc aaaaggatca taaggcttcc tttttccagt 19980 atgtttttcc tcctttttga aaactgggcc agttagctat ctccattttt atttcatgaa 20040 tacatcccca gcgcctggta tatagtagat atggaacatt acactttgga gatattgcac 20100 ccattctcca gtttctccaa agttactaac aatggttcca tcactgtgcc aacatatttt 20160 cttttttcaa tatattggga aataattctc ccagtctgaa aatctgaaca catttcatgt 20220 gacttggtat cctcatatgt cttgggcttc caattctcca ttcctagttt caagttcatg 20280 aactgtaaaa caaaggatta gactaaatct ctaaagttct atccagatgc caaattcttt 20340 tctctttcca tgatacctaa gatagatgcc aaatattgtc ttttacctgg tgtttgtgaa 20400 catgacatca cattacagga gtagcagata ctaaactctc actctgtaaa acactgactg 20460 agttccatga gccagatact gaagtgagct tgttcacata tgttctcatt taatgctcat 20520 aaccctgtga agctgggaat tgctgggaca ttttatttat ttatttattg agacggagtc 20580 tggctctgtc acctaggctg gtgtgcaatg gcatgatctt ggctcaccgc aacctccgcc 20640 tcccgggttc aagcgattct cttgcctcag cctccgcagt agctgggatt acggggcaca 20700 caccaccaca tccagctaat tttgtatttt tagcagagat ggagtttctc catgttggcc 20760 aggttggtca cgaacacttg acctcaagtg atctgcctgc ctcagcctcc caaagtgctg 20820 ggattacagg catgagccac catgcctgcc cgggaccctt gttttagaag gatgactgct 20880 gctataatgt agaaagtgat ttggaagagg ggaggagtgg ggcacgaaag atggttagta 20940 gatgggggtg gtaatgctta cctttcagta tttggaggct tcggagtcct caaaaattct 21000 cttccttgat tggagtcctc ccagccaata gagggcttca cacaaacagt ttcttgggtt 21060 ttgaattgtt tgaccagagc tttcttccga caaaaggttg gggtgattca ttcacttacc 21120 acaccttgcc tgaacattca cttggggctg ccggttatga aggctattgt tctccagcct 21180 gtcacagacg ctttgaagac ctgtgcctca gctggttcta aggagtcagt ttgttcagct 21240 ccgtgccagg tttccaactt atgaaatgtg ctggagatta acacctctcc tgccatttta 21300 tccctactat aattgccagt caaaggattc ctgcagttgc ctctggcagc cataactgat 21360 gaatgttctg ccagctgctc tgaggaccta gaagagcagt tttctatcca ggaccagttt 21420 ccaagggtgg gagggtgaaa tatatcctcc agtgtgacat ttcatctccc agtgatgggt 21480 ggcttgggcc ctttgaagtt ggctctgagg aaccacacac ttgggtctga gcagccagca 21540 gcttatcaca tctggtgatc aatccttcaa aggttcctcc tgaagtctga atttttggag 21600 gtcaaatgga ttccacctgg gaggggcttc tgcttcaact caggacatgg ggagaaggct 21660 gttcctcttc cagggggagg cagttttcat ggcattgaga tgtcctctca cttattcccc 21720 acccacccac caagtccttt gtaagaggag tagggggaga ggagagcgcc tgcagcctcc 21780 tgctcacatt cctagacacc gactcactga gcccgtcgcc gctggaacag cagagctgtg 21840 tgaaatgtca agaggagtta tgctcatagg ctccctggcc tcagtctctt tgtggcttgc 21900 atattcttcc attagtactg tgttcatcac atggaaatca gagggtacaa ttaaaagata 21960 atttgctagt cccagactta atttggggcc cccttcttgc ctgattgaat tacaggggaa 22020 cataatagat ttttggtgag aaatagttgt ctgtgtggct gggagaaaga ttgctcccag 22080 ctctccagct gggcagccct ttcagtatcc cgtatgttat ttccccactt ccagcccacc 22140 tcacctcctc tgtggccctt gtgtgtcccc tcggctagga tcctgacctc ctgctcaaga 22200 gtttaaactc aacttgagac ccaaggaaaa tagagagccc tctgcaacct cataggggtg 22260 aaaaatgttg atgctgggag ctatttagag acctaaccaa ggcccagaca gagagagtga 22320 cttgctaaag gccacatagc tagcccacag tagttgtaac aatagtctta atgatattaa 22380 tggctaacat ttatcaacct ttaatgtgtc ccagactttg tgccaagggc ttacatgcag 22440 tgcattgtcg cattcaaacc cagacagtct ggctctgggc ccaggctgag ctttggtata 22500 gcatggtaga acgttgtcta taatgtctag tctgggttca aatcctggct tcacttctca 22560 catttacagc tgagtgacct caggcaagtg atttaacctc cctgtacctc agttgcttta 22620 tctgtaaaga gaaaaatcac agcactgtgg aatagtgggg gttaaaattc attcatacaa 22680 gtagtgctgc aagcaatgtt taatacaggg tgagcacctg ttcagtgctt ccttcttctg 22740 gctgcctctg gggctagagt gtggtgtctt cgtggtatag atagatagat atggctgagc 22800 tctgcacaaa caccaagagc tgttcttcac tattagaggt agtaaacaga gtggttgagc 22860 tctgtggttc tagaacagag gccggcaagc tatggcccat tgcctatttt aatacggcct 22920 gtgattgatt gatttttttt ttctttttga gacagagttt cactcttgtt gcccaggctg 22980 gaatgcaatg gcacgaactc agctcaccgc aacctctgcc tcctgggttc aagcgattct 23040 cctgtctcag cctctcgagt agctgggatt acaggcatgt gccaccacgc ctggctaatt 23100 tttgtatttt tagtagagac agggtttctc catgttggtc aggctagtct cgaacttcca 23160 acctcaggtg atctgcccgc ctcagccttc caaagtgctg ggattacagg cgtgagccac 23220 catgactggc ctgattgact gattttttta gtagagatag ggtcttggtt tgttacccag 23280 gctggtctca aacttctggc ttcaagcagt cctccctcct tggcctctcg aatgctggga 23340 ttataggcat gagccactat gcctggccta tatgacctgt gatttttaat ggttagggga 23400 aaaaaagcaa aagaatgctt tgtgacatgt ggaaattaca tgaaactcaa atatcagtgt 23460 cccagcctgg gcaacaaagt gagaccctgt ctctacaaaa aataaaaaaa aataagccag 23520 ggccgggcgc agtggctcac acctataatc tcagcacttt gggaggccga ggcaagtgga 23580 tcacctgagg tcaggagttc aagaccagcc tgaccaatat ggtgaaaccc tgtctgtact 23640 aaaaacacaa aaattagccg agcatggtgg catgcgcctg tagtcccagc tacttgggag 23700 gctgagacaa gagaattgct tgaacctggg aggcggaggt tgcagtgagc caagatcgcg 23760 acactacact gcagcctggg caacagagcg agactccgac acacgcacgc acgcacacac 23820 acacacacac acacacacac acgctgggta tggtggccag cacgtgtggt cccaggatgc 23880 actggaggct taggtaggag gatcacttga gcttaggtgg ttgagactac aatgaaccat 23940 gtttatacca ctgcacttta gccagggcaa cagtgtgaga ctgaatctca aaagaaaaaa 24000 aaaaaaaaga aaaaaatctt tccataagta aatatctgtt ggaacatagc catgtccctt 24060 agtttatgtt ttatatatgg ctgcttttgc cctataatga cacaattgag tggccacgac 24120 agtctgtatg gcctgcagag cctaagatat ttgctctctg gccctttaca gaaaaagtgc 24180 cttgacctgt gctctagagc catatgtacc aggtttgaaa ctcagcctca cagctgggtg 24240 tgatggcacg catctgtagt cccagctact ctggaggctg aggtgagagg atcacttgag 24300 tccagaaggt cgaggtcaag attgtagtga gccatgatgg catcaccgca ctccagcctg 24360 agtgacagag agagaccctg actcaaaaaa aaaaaaacaa aaaaaaaaaa caccctcacc 24420 acttatcagc tatttgtctt gagaatagtg acataacccc tcagaaccta tttcctaatc 24480 tgttaaatga ggctgatgac gtttcctcct tttactggca atttaaacat gatggataat 24540 aaatgctaag cacttaacac agggcctaga agatattaac tgctcaataa atggtagctt 24600 cttaacagta ttcaaaccca tgtgctctta tcacatgcat tgttgtccct gtgtccagtt 24660 ggtggaatgg gaaaaggctc ccttgtaacc ccatctacca tctttatcag actttcctgc 24720 catggttcac agtaagagat agaagctgca cggtgacttc tggctcttta caatggtgag 24780 cggtgtgtgc ctggtaaggg agagctgatg tcactgcccc aaatccagta gtgagatctg 24840 agtgttctgg tttcctccag cagccttgct ttttccttta caatcctgca ggcagggaga 24900 caagggcttt ctacatggta ggctctggtt tggtcatcgt cacaactggg ggctgttcag 24960 gtgggctccc attccagata cctaggctta tcaatccctt ttggcacccc aggccttttt 25020 ctccctcatg ccccattttt cagtttgaaa agcatggtta tcacaggaca agtagaagaa 25080 gctccactgt ccactgaggc caatggatgg tgttctgcat gtgaacactc agtgaatagt 25140 gagtgaatga gagtaacctg ggctccatcc tatttgcaga gagctttgga aaagattttt 25200 ctccttaaag agccagaatg aagcctggta gtgggagagc tccagctcta gagtcacatg 25260 agcctacatt taaattccag ccctgccact gactcccttt ttgaccttga gtgagttacc 25320 taatctctct gtacctcact tttcttgtct gtagagtggg aataattcct gtctcagaga 25380 aataaaagag tgcatatagt gtttgccaca tggagacaca tcaggtgtag gttaatactc 25440 tgggccttgt ttccttattt gcaacacagc cctgccctgg agtggaagtg gcacctccca 25500 ttggtcagct cttgaggctg tccccaggac aggcagaggg agggaatgaa tgggagccct 25560 agtgccagga cagaacagat ggcagctcag agctaggatg gctctctgga cctgtctctc 25620 ctaccagagg tccccccgtc tggtgtggct cttcctggac ctggcatcct ctgctttttt 25680 tttttttcca cctccaagca gaattactgt cctgtaggca gctcctctgc ttgaggacat 25740 ctggggccag atatgttcac actctatcct gccttgccct tccctgagct caggatggac 25800 gctcaattgg tcccagttat tgtctgcagc gcctgcctgc agcctcgatc cagcccagct 25860 ccaccccttg cctgcaaggt ctgtttccta acagctgctc caaccacaca cctcggttct 25920 gcgggagccc ctcctcttcc tccctccctc cctcattcag gggtgggact gaagaagaag 25980 gctaacttga cagcagcgct tctttcttag ctagtcaccg gcccctgctc aagaatgcca 26040 gtgtgtgtgt agcctccaca gagaggtcgt tttctcggag tccagagggg ccgcctgagc 26100 ttctgagaac tagggaggag ccatcccagc catgagcccc tgtgggaatc tgctgggggc 26160 caagtggcct ggagtcctca ggctcccgca gctgctccgg agggagaggt gagctcaggg 26220 cagcctgcct gcagccagag gtgccgggag ccccgggcct gtcatggtgg ccatctacag 26280 ccggcctgag gcagtcacag acggatttgc agctgagcct gtctatctgg tgtgggaaga 26340 agatggggag ttacttgtca gtcccggctt acttcacctc cagagacctg tttcggtgag 26400 ttggtctccg agttcccctc tccatctctc ctggcccctg gtcctgagag gagggtggtc 26460 tccctaaatc tccttctcac ttagtccttt accatcggtt ctgccgggca gaagccagcg 26520 gaggttatac ccaaggagaa tcggccttgt gaggtacccc cattatgtcc tggaagtggt 26580 gaggggaggg atatacccag aaggaacttc ttagggagct ccagctcccc ttctatccca 26640 gacaaacctg aaggagcctc caaaagatgc cactgacctg cccattgtag atgttactgc 26700 ttccgggggg aatagcccaa atagagtgct gtttccagct ctcacatgtc ttacctgcgg 26760 gccatgctgc ctgcccagga atttgtccca acaagcagga tgggcaggtt ttgccaaact 26820 gtggaaactg gcaagtcctg ggtgtgggta gcctggtaca cagtaggcac cttataaacg 26880 tttgttctct taatggcagg cacatttgcc tctggccttg aagggcttct gagctcccag 26940 gtgaatgtag ttgctgggga aagacctggg cgagtgcttc taagactgga gcaatgggct 27000 ttagagtgtt cctgagctgc tgggccagcc cccacacctc ctcagtccct aggcctaagt 27060 acctccacga gcctctctct gtggggcttc tcagagggag atgtggaaac tctacctcta 27120 acctggcttt ctttgctcat tgccccactc cacctcccat agaaactccc cagggggttt 27180 ctggccctct gggtcccttc tgaatggagc cattccaggc tagggtgggg tttgttttca 27240 ttctttggga gcagcctgtt gttccaaaaa ggctgcctcc ccctcaccag tggtcctggt 27300 cgacttttcc cttctggctt ctctaagcta ggtccagtgc ccagatcttg ctgccgggat 27360 actagtcagg tggccaggcc ctgggcagaa aagcagtgta ccatgtggtt ttgtggaatg 27420 accggaccct ggtagattgc tgggaagtgt ctggacaggg ggaaggggga agggaactgg 27480 tcctcaatgc tgactctacc aagcgccctg ctagacactt tatcctttaa tctctcaaca 27540 gcctaaagag attatatatc cccattttac agatgaggca accagtttca acagagttaa 27600 catatggagc ctcactgggc agctttttct gtcttcctga ctttctctca tccttcaggg 27660 ggctgcaggt ttgttttctt ctcctagtgg agaggaaatt ctcaggtttg ttttcctctc 27720 ctagcagaga gtaaaaaaag ggatagtttg cctgacttgt tgaaggtgtg gctgagattg 27780 ttttctaaag agccaatgga aattgatctt gagtttagga gaaagctttt acatgtggaa 27840 ttaagatgcc aagtgttgaa gtagccacat ttcaggtcct cattaatttc tcttaatcct 27900 gggaaggcag cttaggagaa gggttgttcc tttaggagcc aggaactata ccccttttac 27960 ccttggagag gcagggaagc cagggaggac acaacttctc aggaagagga gaagctagag 28020 cagatagtga actctcaacc tgaaccttta agggccagac cactaatgcc acccaagtcc 28080 acctgccgtt tgtcttgttc tgtcccaggc tttctggaga acctgatctt cttgccccta 28140 cccccaagct ccgtttgccc agctagagtc tggggggtac tgactgactt tcgtagacat 28200 tcttcccttc cccaaataag aggccacatt cctgaagtca cttctgaaga gatagctgcc 28260 acacagggct ctttcccccc agggagggac cacccagacc ctctgctctc ccaggtatcc 28320 gttaccacat cactacctgg tcagaaagct gtttctgcca ttagcccctc cctcttttat 28380 tataggatat cctcaagggc tcctctttgg gcctcagttt catccttggc agaaagtaga 28440 agctagactt cttgggctcc tgaacagggt ccttgctgga ttctgtgaaa caaattaagt 28500 tcttgaccct aggcctctgg gggagtacaa agtctatggg agttctgggg ctgtggttgc 28560 aaggaaagtg acgcaaccag attccatggg gacatgatca ggcgtgacat gtgagggagg 28620 aagagggagc aagggaatga agaatacaac ttctgtgtcc catacacccc tgcctgacag 28680 gccatacata ctcagcagag aatgcactgt ctttcctacc acactagcgt gaggagtgag 28740 ctgcaattac cactgtgctt ccaagtaaga aaatacctca aattggaatt tacaaaagag 28800 gtaaattagg gagtggcttt tgtcggacat ctttaaagca tttttctttt tatagaattt 28860 cacttaatgt ccaatactga tttaatgagc ttgggtttac acattatctc ttgaagaaaa 28920 caaatgaacc tttgtgttcc aaagcaatcc atgtttaaag ggaaaaaatt atgcataact 28980 ctgcccagct tcacagtaac ctttggcagg tgccttaggt cctctgggac tcttttcctt 29040 atctgaaaaa tgaaggactt ggatcaggtg aatggttccc agctctgcaa cttatgtggc 29100 tcctcagagg cacacaagct cttttccatt atttgccaaa taatggaggc cctgtcttta 29160 actgcagtac aactacacaa aatacttgaa actacagtct tcctggtttt tggttggaac 29220 tgaatcagtg cactctagca acacttattt cttgctgttc gtaggcttca ttatgtgttt 29280 ggttaatttt ttaaaacaac aataacatat tccataataa ttacagctta attggcagac 29340 tgtttcagtc tataggatct gcaggaagga ggagtaataa agggattttt gactgagctc 29400 ttatggaaca gagtctctct aggcccctgt catatctgcc cttctgggcc ctggggaaaa 29460 gttggcatcc ccagttgtgg tgctctccag gtgccctcag gctgtggtgg agggagcttc 29520 ccattctctc cttcagccca ctcaattcag aggctagggg ctgaaagaag cttctctaca 29580 actggctgtt cactgggagg ttaagggatg accatccagc caggccttcc tcaggacatg 29640 ggagggctta tgctttaaca tgtgtaaatc cactgcaata atgactggtt cttttacccc 29700 ataaggttga gaatttacct gtaaacattt ttgtctgaag aatttggatg taagtgaggg 29760 ctgggcctct atcttatctc acttggcttc tctcagcaca gcaccttgcc tgcttgttct 29820 tacacatcct agatgcacag taactatttc ctaattatta gaaatctatt agaatcaatt 29880 gatttcagct gggcttggtg gctccttcct gtaatcccag cactttggga ggctaaggct 29940 ggaggatcac ctgagtccag gagtttaaga ccagcctggg caacataggg agaccctgtc 30000 tctacaaaaa ataaaaaatt agccaggcat ggtggtgtgc acctgtagtc ccagctactc 30060 aggaggctga ggcaggagga tctcttgagc ctgggaggtc agactacagt gagcaatgat 30120 tgtgccactg cactccagcc tgggtgacag agtaagactc tgtctcttaa aaaaaaaaaa 30180 aaaaaagttg atttctattt ggatagataa ataattcatt ttaggacctt tctttttcac 30240 ttacagaaat ctgtttcatt ctgggctgag aagcaggtcc atattgctag gcataggaga 30300 aaaaggggtc tgtctgcatt tgcccttggt ggtctcaaat tggggaggga aagaaatgaa 30360 cacttactgg ctaccttctg tgagccaggc atcatgcaag acatctgtac ataatttaat 30420 tctcataacc ccataagata ttattagcaa tgtacaagtg aggaaactga ggctcagagt 30480 catgaagtaa ctggccttgg gtgacacaga tggtaaatgg cagagaagga atatggatcc 30540 aggtcttgaa agagaaaatc tcaactgatt atctttttta aaaaactcat atgttctctg 30600 ctgactcaaa aggtctctgt gtggatctgg gttgacccac tgaactgacc atcagggttc 30660 catgcacttt gtatctgccc aagccctcag aacccctcag taatgttttg gaagatgagt 30720 tttggaggtt gtccttaggc atagcctcag cgtatgtagg cctctaggtg atctccccta 30780 acctgaggat ttcagctcaa ttcactctgg ctcctcagga cagtgggatg actggttcag 30840 acctcagctt taccacctcc cagctgggta ctcttctacc tacagccagg gcagattttg 30900 actttcactt gaaacttcca aaaattgaaa ggtagaaaaa cagccttggc tttgggaaga 30960 acgtatgatg tccatggcct ctaagcatct gaggtgggac atgttcgagt agcaccttac 31020 agttccaaag tgtgttctgg gttctttgtt taaaagaaca gagactgctg gggaattgaa 31080 cactgtgaag tatatgaagg aggagaattg tgctatttaa cattcagtac ttgggctaaa 31140 ggagaagcat cacgaagtgt taacactcaa agggtcttga gctgtcaggg ctccagcttc 31200 cttattttca caggtgagaa tcctgaggct cagctgttga gatgtgctgt ctcactccgg 31260 tgacatagta cagtggatgt ggctttgcag ccaagcacac atagcttcac attccagctc 31320 catcaattat gtattgggca gctttgcaga atgatttgac tttaactctg cttttcagtc 31380 ttctgtaaaa cagggataat cctgctaccg tagggttgtc aggattagag ataatataaa 31440 taaggtacct catataggac ctggattatg gctggcattc aataaatagt agctgttaat 31500 tgatagctaa gctagaactc tgaagtctac catggcaact tcttaagtgg tctgagaacc 31560 cagttgtgtt ctgtggcaaa acacagctta gggatccata cccagccctc ctgtcagctg 31620 ttcaccttcc agttcttcag agacatgtgt ggcagtgact ttggccacat agctggctgt 31680 gccctttaaa ggcattcctt gacacagata tgtggactgg tgacgttgct ctccagccag 31740 gtgttcttcc cagcaggctg gcctggctgt ctcctgcatg cctgtacttg tttgtctccc 31800 tgctccctct cctgggcctg gccagagcta cttgcagcaa acaaaagcag gatattggca 31860 atggaaagga gggtgtgttc tggtgctccc atgccctgcg gcgcacatac cattgcaagg 31920 gcgtaacaga gcccaggcct gcatttgggt gcaaataagt ctgcacacag aagaaaagaa 31980 ggacctggtg accaggagcc atggaaccct tgtgctcccc tacctgggct actggttctt 32040 gccactccta ccattttcag tttggaaata tttgttaagg ctttgctctt ccaggtcctt 32100 tgcttggtgc tgagtctacc aagagtaagt gggatgctgt ttttgtcctc agggagctaa 32160 cagtctagtg aagaagaaag atggttgccc aggaacttct aagtcagaag gcaggaggca 32220 agaaggaagc ccctgctcct actgccagcc ctctgttggg caccccatag ttcttcagaa 32280 ccacatttaa tcctcactgc aggccaggca tagtggctca cacctgtaat cgcagcactt 32340 cgggaggcca aggcgggcag atcacttgag gtcgggagtt cgagaccagc ctcaccaaca 32400 tggggaaacc ccgtctctac taaaaataga aaaattagcc gggtgtggtg gcatgcgcca 32460 gtaatcccag ctactcagga ggctgaggtg ggaaaatcac ttgaactcgg gaagcagagg 32520 ttgcagtgag ccgagattgt gccactgcac tccagcctgg gcgataagag caaaattcca 32580 tctcaaaaaa aaaaagaaaa aagaaaaaat cctcactgct accttgaaag taggtgatga 32640 cattgccatt tcacaaatga gaagtgaagg ggctagccca agatcactta ggtggtaaat 32700 ggtggtgcta agattagaac ctcagatcat ctagggaaaa acacagatat gcacagagtt 32760 aaggggaccc agggtattgt ttgtcctctt gtttcacagg tggggaaaca acccagagag 32820 ggaaaggggc ttgtccaagg caatttagca cccaagaact tgaacccata tctctctcct 32880 cctcatttag agctcatccc acatgtatct tatattgaga ggagtgtgag ccacatacca 32940 agaacagtct tcccctctgc ctccaacctc actgtgcagt tttgagacac ttcacagcca 33000 tactcttcat gccataccca gcccttaaga ccctgaagtt ccccttccat aagacaagta 33060 ggaaaagcta tagggtaaaa atagccatca gtgtttgttg agcacccagg aggaattggg 33120 cactccagaa agataaaggg attctcaggg acttgcttct ctagacttcc ctagctcagc 33180 tgcttcaact cattcctgcc cctcttctct acctcccgca gtgctcagaa gtagtagaac 33240 tcactgtggc ctctcacctt gcattgttga gttttattta gactttctct tcctcaactc 33300 ttcataagct catgaaaggt gaagtagggt gccctgtgta tttatctttt atatctgcag 33360 tgcttagcaa gttataataa tgcacttgcc tggcaaaagg ctttctctca tacattagct 33420 tatttcctct tcacattggc tctttgtagt aataggatgc tattagttat tttcaatgag 33480 agaaagctac taagagaagt tgtccagcta gtgacagtaa gtggctgata aagtgagctg 33540 ccattacatt gtcatcatct ttaatagaag ttaacacata ctgagtttct actatattgg 33600 gtcttttttt tttttttttt ttttttttta gagacggaat cttgctctgt tgtccaggct 33660 ggaacgcagt ggtgcaattt tgggtcacca caacctccgc ttcccaggtt caagcgattc 33720 tcctgcctca gcctcctgag tagctgggac taccagtgca cgccaccacg cccggctaat 33780 ttttgtattt ttagtagaga cagggtttca ccatgttggc caggctggtc ttgaactcct 33840 gaccttgtga tctgcccgcc tcagcctccc aaagtgctgg gattacaggt gtgagccacc 33900 gcgccctgcc tatattagga cttttatata agctatctct agctagctag ctagctagct 33960 ataatgtttt ttgagacaga gtctgactct gtcacccagg ctggagtgca gtggcgtgat 34020 ctcgactcac tgcaacctcc acctcctggg ttccagtgat tctcctgcct cagcctcccg 34080 agtagctggg attataggtg catgccacca cgcccagcta attttttgta tttttagtag 34140 accaggtttc accatgttgg ccaggctggt ctcgaactcc tgacttcaag tgatccaccc 34200 gcctcggcct cccaaagtgc tgggattata agcataagcc actgtgccca gctgctctct 34260 atatttttaa tacatattat ttccattaat tttcacagca gttcatttta tagatgagga 34320 aactaggcca gagaagtaaa atatcttgcc caagatgatg taactagtaa gtggcaggat 34380 caagattcaa accaagcaat gttcaaacct cttggaagca agaatgtggc cactgtggaa 34440 ggtgcaaggc cttgacaaca agaataggga aaagaaggaa ctagaaggaa agagatggca 34500 tgggctcagc aggccaggga gctcttagct gtgtgtgttg ggaagctcag aagggaggaa 34560 gaggttgtct gtgcaggtaa gtcctgagaa cacaccagac ttttgagagg tggagcttca 34620 tagccaggtc attaggggag aagggagcta tagatttttt tttttttttt tttttttttt 34680 ttttttttag agacggggtc ttactatgtt gcccaggctg gtcttgaact cctgggctca 34740 agtgatcctc ccacctcagc ctcccaaagt gctgggatta gaggcatcag ccaccccgcc 34800 cagcgagcta tggatctaac atgtacatct tacacagtgc taatagaatg ttgggtttct 34860 tccccaatat tttattttga aaaaaaattc aaatatatag aaaagttgaa aaatgtagtt 34920 caaagaacac ctacatacct ttcacataga ttcatgattt gttaatgtta tgccactttg 34980 tatatatctc tctccctcct atctgtatac ttttatttat ttatttttgc tgaactattt 35040 cagagtaact taaaggcatc ttgattttac ccttgaacag ttcaatatgt ttctgctaag 35100 aattctccta tataagtcag atatcattac atctaagaaa attcacggca attttacaat 35160 ataatattat agtccaaatc catatttcct cagttgttcc aaaaaatgtt catggctgtt 35220 tcctttttta atctaaattt gaatccaagt ttgaggcatt gtatttggtt gctgtgtctc 35280 tagggttttt aaaatctgtg ccttttcttc tccccatgac tttttagaag agtcaagacc 35340 ggttattctt atagaataac ccacattcta gatttgcctg attagttttt ttatacttaa 35400 cgtatttttg gcaagaacat tacattggta acgctgttgg tgatgggtca gttttgaaga 35460 gtggagatga ttaaactgct tttgttcatt gaagtatctg tcaagaccag agatccttaa 35520 ctggtgccat aaataggttt cagagaatcc tttatatata caccctgtcc cccacctaaa 35580 ttatatacac atcttcttta tatattcatt tttctagggg aggcttcttg gcttttatca 35640 aattctcaga gggccccaag acccaaagag gttatgaaac actagtctgt ccactgaggc 35700 aggcaacaca gagctggttt ctggggcctt gttcagtctg aaccagcttc ccttggggag 35760 atagcacaag gctgtaactt tgccccatct tggctttgga tcaaagagga ctgtccattt 35820 tgttgtcata cctaggaacc agggacagct tatgtggcct ggttccaggg atccaggaga 35880 atttcagttc ttgtcttgcc tttcaggtgt tcagaatgcc aggattccct caccaactgg 35940 tactatgaga aggatgggaa gctctactgc cccaaggact actgggggaa gtttggggag 36000 ttctgtcatg ggtgctccct gctgatgaca gggcctttta tggtgagtga atcccttcat 36060 atctgcccct cttggtcttc agagtccatt gacagtgctt ccagttccct gtggcctgtt 36120 aatcttttag tctttccatc agccagggca tctcccttta tttattcatt cattcaacta 36180 gcaggtatca attgagcacc tactaagtga aaggtaagat ccttccctca aagacttaat 36240 agttgaacgt tgggagtggg aggagaggca ggcagagagg agacacaata tagttggata 36300 aggacctcca aggagagtgt tacaggctga gaggaggata tacttaggtt gtctttaggg 36360 aatcagaaaa ggagactctg gaataggctg gcagagagag gggctacctc ctatacctgc 36420 tctggacaaa cgactttaag catagtgaca gatttgccaa ccctgtattg gaagaactga 36480 tcttttttag tggggatgat tacttctggg gatttcttct cataactgag accaaaacag 36540 ttttgtgcag tctcagaaat gacaggaggt accaatctga cacttccttt ggaagctcta 36600 gggcagagag tgaaagagtg gattttgacg ggggccttgc ttggaggtca ttcacccacc 36660 cctgtcctca ctccagcaac agtgataact cacttccttc ctccctttgt acacccttct 36720 ccccacctgc tcacaggtgg ctggggagtt caagtaccac ccagagtgct ttgcctgtat 36780 gagctgcaag gtgatcattg aggatgggga tgcatatgca ctggtgcagc atgccaccct 36840 ctactggtaa gatagtggtc ctttgtctat cctctcccat ataagagtgg ctggcgggga 36900 gggacagtgg cagggtgagt tgggcagaag gagtgttagg gtagtcagag cattggattc 36960 ttaccacagc agtgctctta accagctctt taacttgtaa gcagaatgat ttacacatgt 37020 ctctaccctt tttccttacc aaccttgaaa atgtcttcac tctgccctgc aatcctccca 37080 gtgggaggca ctcttcaagg acgatcccag aacattaaag tcaaagaccc cttagagctc 37140 accctgtcca accaccttgg ttgataaaag aagtcagcct ggggcccatg gaatagaata 37200 gtacaagggc aaggttctca ttgtgagtca aaggtagagt gaagagaacc cagaccatct 37260 caccccaacc caggccagtg tttttccaaa tataccactt gctgcagatc tagctcagca 37320 cccccagtcc cagcccaccc tgagaaccca ggctcctcat tctgagcagc cagctagaat 37380 catgacaaag agggtggtag tgagactatg ggtactgttg cttaaagcca catggtgcag 37440 tggttgctgg ggggcttctg tgtgggactc tagcatctta ttcccccctg tgccctctcc 37500 ccagtgggaa gtgccacaat gaggtggtgc tggcacccat gtttgagaga ctctccacag 37560 agtctgttca ggagcagctg ccctactctg tcacgctcat ctccatgccg gccaccactg 37620 aaggcaggcg gggcttctcc gtgtccgtgg agagtgcctg ctccaactac gccaccactg 37680 tgcaagtgaa agagtaagta ttttgagaac ccttcagcag gggttcttga gcagagtctg 37740 taaatgggcc tcagagggct tagacctcca aagtctcatg cagaactccc tttattctca 37800 tctcatatct ttctcctgga ccccactatg ctgtaaccgt acctgggcct tggcacttac 37860 tgttctctct gcccaggcta cttcctaccc gatacttaag gcaagaatca ctcacctttc 37920 aggtgtcagg tttcaggtca tgtttgctct ttgaaatcat ctggcttgat tatgtgtatt 37980 agttgtttat cttctatccc ctccactaga atgtaaattc cagaagaaac ttgctgtctt 38040 attcagtgct gcatgcccag ggcttggaag agtacctggc atatagtagg agttgattga 38100 ttattatttt gtcagtcgag agaatgaatg gagaaaatgt ggtccatggc ccaaaagaag 38160 ttaagaccct atcctagatt caggccagag accagatgga gaaagagtct gtgtctatct 38220 aataccagta atgtcgtacc tctggccgct taccatgtaa atattgattg tgtatctacc 38280 atgtgttgga cactaggcta gtgcttgcac agcaggtgaa agatactaga gtttgggaag 38340 tcaggaggag ctaaggtctg ttctacaacc ttattagatg aagaggagag ggaattgtgt 38400 tcagggcaga gggagaagca tttctccaaa agtaggagtc ttaatcatgt ctgatgtagg 38460 ttgagtgtgg ccagaaaagg ggctgttaag tatagagggc ctggattatg aaaatccagc 38520 agatccattg agagtttaag cagcaaggtg ttgtgaccaa gttaacattt tagaaggatc 38580 actggtatgg aggttggatt ggagagggga aagcctaaag gtatagagac tagttaggaa 38640 gctattgtag gctgggcatg gtggttcatg cctgtaatct cagcactttg ggaggctgag 38700 gtgggaggat tgcttgaggc caggagttga agaccaacct ggccaacata gcaagacccc 38760 gtctctgttt ttcttaatta aaagaaaagt ccagacgtag acatagtggc tcacgcctgt 38820 aatgccagca ctttgggagg ccaaggtggg cagattgctt gaggtcaaga gtttgggatt 38880 aggccaggcg cagtggctca cgcctgtaat cccagcactt tgggaggccg aggtgggcgg 38940 atcacaaggt caggagatca agaccatcct ggctaacaca atgaaacccc gtctctacta 39000 aaagtacaaa aattagccgg gcatggtggc ggacgcctgt agtcccagct actcgggagg 39060 ctgaggcagg agaatggcgt gaacctagga ggcggagctt gctgtgagca gagatcacgc 39120 cactgcactc cagcctgagc gacagagcga gactccatct caaaaaaaaa aaagagtttg 39180 ggattagcct ggccaacatg gcaaaacccc atctctacaa aaagtacaaa aaaattagct 39240 gggtatggtg gtgcgcgcct gtaatcccag ttactcagga ggctgaggca tgagaattgc 39300 ttgagcctgg gaggtggagg ttgcagtgag cccagatcat gccactgcac tccagcctgg 39360 atgacagagt aagatgccat ctcaaataaa aattaaaaac aaagtttaaa aaaaaaatag 39420 aagctattac cgtgatccag gtaagagatg tgaataacta caatgatgga aagaaggcag 39480 agttcttaga gatgggagta ggagagatga gggaactcca gattgggaag atgatgttca 39540 agtttctggc ttaggccaca gggtgagtgg caattccctt cactgagatg gggcatcctg 39600 gaaaaggtgt tgcctttctg tgtgggtatc ctgggcccct taggggccac tggtggcctg 39660 ggacctggta aaccttccct gcacaagcag aattggtcaa gcaggttttt aggacatctt 39720 taccctgcct caactcttgt ctggcccagg gtcaaccgga tgcacatcag tcccaacaat 39780 cgaaacgcca tccaccctgg ggaccgcatc ctggagatca atgggacccc cgtccgcaca 39840 cttcgagtgg aggaggtaga gtgtgtgtct aatctgtctt gtgagggtgg gacatggaac 39900 agatcctctg ggaaatcagg ctgtagcctt taccttttcc tacccccagc ccatctcttt 39960 gtcttagcat tgagcctgtg accactggtg acctatttca gcgtaacagg ttcccagggt 40020 agcagggatg gttgatggac gggagagctg acaggatgcc aggcagaggg cactgtgagg 40080 ccactggcag ctaaaggcca ccattagaca agttgagcac tggccacact gtgcctgagt 40140 catctgggtt ggccatgggt ggcctgggat ggggcagcct gtgggagctt tatactgctc 40200 ttggccacag gtggaggatg caattagcca gacgagccag acacttcagc tgttgattga 40260 acatgacccc gtctcccaac gcctggacca gctgcggctg gaggcccggc tcgctcctca 40320 catgcagaat gccggacacc cccacgccct cagcaccctg gacaccaagg agaatctgga 40380 ggggacactg aggagacgtt ccctaaggtg ccacctccca ccctggctct gttctgtcct 40440 atgtctgtct ctcggatgaa gctgagctgg ctttcagaag cctgcagagt taggaaagga 40500 accagctggc cagggacaga ctatgaggat tgtgctgacc cagctgcccc tgtggggatc 40560 acagtttaca gccagagcct gtgcggaccc agctgtctgc caggtttcct tagaaacctg 40620 agagtcagtc tctgtccact gaactcctaa gctggacagg aggcagtgat gctaaaccct 40680 gaagggcaac atggcctatg gagaaagcat ggagctcaga gcctggagta cgggcacaga 40740 taggattgaa taaattgtgt agaaagactt tgaaaacaat aaagcaaaag atgaatgaac 40800 gtttttttta gacttgaggg accaacaacc cccaaacccc agattctgcc aggtccatgg 40860 ggaaggagaa gttgccttga gtggaagccc caagtaggga gacttacaga aaagaagtca 40920 agagcactgg ctcccaggca gaaatactga taccctactg gggcttcagg ctgagctcct 40980 cccttcacaa atcacttcat ctctctgagc ctgtttctgc atctgtgaca taagatggta 41040 agataaaggt ggctgtctca ccaattatgt aaggattaaa tgtggaaaag gacataaagt 41100 tgtatagtgc tgccataggg acagtgttca gtaaacgtga cacattctta gtatcactaa 41160 gaatcaggtt cttggccagg caccgtggct catgcctgta atcccaacac tctgggaggc 41220 ctaggtcgga ggatggcttg aacacaggag tttgagacca gcctgagcaa catagtgaga 41280 cactgtctct acaaaaaaaa aataataata ataattgttt ttaattagat gggcagggca 41340 ctgtggctca cacctgtaat cccagcactt tgggaggcca aggccggagg attgcttgag 41400 gccaggagtt caggagcagc ctgggccaca ttcctgtctc tacaaagaat aaaaaagtta 41460 actgggcatg gtggcacatg cctgtaatcc cagctactca agaggctgag gaggaggatt 41520 gcctgagccc aggagttcaa gactgcagtg agccttgatc acaccactgt actacagctt 41580 gggcaacaga gtgagacctt gtctccaaaa aaaaaagttt gttttttttt atccactctc 41640 ctcaccaaac aaactgagta agttagagcc ctctcagctg gcatgtgttg gaaacagtgc 41700 cctctcatta aagtgctgcc ctcactccca ttgcctcttg gccttggtca gtatgatgaa 41760 attagtggga ggcagggcaa cagagggcag ggaagagcta gaaatccatg gcctggaaaa 41820 gggaagattt gggagtggcc aggtatctgt agagccacca tgcagaggag gggggcagct 41880 agccttgtgt gctctggtgg gcatggtcag caggaggcag agcaaaagga caagggtaag 41940 taaacctgta ggtcgggaca agccaagagc catccagcgt cagtcctctc tgggtagccc 42000 aagtaaagca ggagcatacc ccagagagaa agttcgcagg gctgttcacc tgcagtgctg 42060 tggacttcaa ccttcttgtt ccttcttcag taagtgaaaa taacagtcat tgaccatgac 42120 tattatcgac cgcttttgaa aatgtaaaca tagtgacttt attgctgtaa aaatcatacg 42180 tgtttatcat cttaaaattc aggaaacatg gacaggtaca aagatgtgca aaatatcatc 42240 caaaatccca tttgctggcc aggcacggtg gctcacgcct gtaatcccag cacattggga 42300 ggccgaggcg ggcaaatcac ttgaggtcag gagtttgaga ccagcctggc caacatggtg 42360 aaaccctatc tctactaaaa atacaataat taggctgggc gcagtggctc acgcctataa 42420 tcccagcact ttgggaggcc gaggtgggcg aatcacaagg tcaggagttt gagactagcc 42480 tggccaatat ggtgaaaccc catctctact aaaaatacaa aaattagggc cgggtgtggt 42540 ggctcacgcc tgtaatccca gcacttaggg aggccgagac agatggatcg cgagatcagg 42600 agttcgagac caacctagcc aacatggtga aaccccatct ctactaaaaa aatacaaaaa 42660 ttattcggtt gtggtggcac acgcctgtaa tcccagctac ttgggaggct gaggcaggag 42720 aatctcttga acctgggagg cagaggttgc agtgagtgga gatcccgccg ttgcactcca 42780 gcctgggcga cagagtgaga ctccatcaaa aaaaaaaaaa aaaaaaaaaa aaattagccg 42840 ggcgtggtgg cgtgcaccta tactcccagc tacttgggag gctgaggcag gagaatcgct 42900 tgaacctgga aggcggaggt cgcagtgagc cgagatcgtg ccattgcact tcagcctggg 42960 cgacagagcg agactctgtc tcaaaaataa taataataac aataactagc cgggcctggt 43020 ggcacatgcc tgtagtccca gttactcagg aggcggaggc atgagactca ggtgaactag 43080 ggagacagag gttgcagtga gccaagatca caccactgca ctccagcctg gttgacagag 43140 cgagactctg tctcaaaaaa aaaaaaatcc catttgctca ttttttggat actagtataa 43200 ctatcactct aaaccagtta gtacttaaat caagcagata tgggagatgg tgaattacca 43260 tctacagtgt tgtcatatat gtcacatact gagcattatc agctagtaga atctagttaa 43320 ttgttctatg tgtgatgtat gcagagttcc cattttgaat gtgtttttac tatgcttaaa 43380 taaatgactg atgtcagcaa ccccaaaatg atacatctga tgtaagagcc cctgttcccc 43440 aataataaca tctaaactat agacattgga atgaacaggt gcccctaagt ttcctccctc 43500 cagggtttct tggccggtct ctgaggacta cacatcccta ctcccgtctt tcctcatctt 43560 caggcgcagt aacagtatct ccaagtcccc tggccccagc tccccaaagg agcccctgct 43620 gttcagccgt gacatcagcc gctcagaatc ccttcgttgt tccagcagct attcacagca 43680 gatcttccgg ccctgtgacc taatccatgg ggaggtcctg gggaagggct tctttgggca 43740 ggctatcaag gtgagcgcag gcaacaattg ctttgctctt ctgcccccag tccctctgtc 43800 actgtctttc ggggatttct catcacttgg ccccacccca caccatgcag gatgccaggc 43860 ctccttcctg gctttgggtg ttggtgtgag aggtatcctt cacccccacc caggccacct 43920 aaggtcaatg ttgctgttac agtgagcttg tggacctgga gatccaggtt gggttgagct 43980 gtgcctgtgg ccctcctgcc tccagtcagt gggtgtttgt taggtgcctg cagacctcag 44040 taccgggcat gctacaagga gcacacaggg gaatggctcc tgcctccctg gtgaacagtc 44100 tcagggacta acctctctct ttctctcctc ctcctcctct tctgctgaga actgggaggg 44160 ggggtcaggt aagacgtgtg tctcagcttg ggggcagcag ggctggagag ctcacccccg 44220 atccacccag ctccctggtg catgtctttg gcactgacct tcctgccccc agacttctgt 44280 tcactcagga gactcacttc tatgccaaat gaccagagcc cctgcttggc ttggcagcat 44340 cccctcctgc cttcttcccc acttcccttt tctgggttct tgcctgtcct ctgtgcatgc 44400 ccagctctcc aggaaagagg gtttgcttcc gtgtgagtcc catgttgctc cacgctgcat 44460 cttccacaca tgaactctgt cattctgacc cggctcagtg tgccctccaa gggatgggat 44520 ggccagctgc atagattttc tcaaacagtt ctccagaact tcctctggtc tcagcaccat 44580 taacagtcac cctccctgta ggtgacacac aaagccacgg gcaaagtgat ggtcatgaaa 44640 gagttaattc gatgtgatga ggagacccag aaaacttttc tgactgaggt aagaagatgg 44700 agggggcccg ggaggttggt gtcaccattg gaagagagaa gaccttacaa ataatggctt 44760 caagagaaaa tacagtttgg aattactgtc ttaaagacta agcagaaaag agccctagag 44820 gaatatccca ctccctctaa attacagcgt aattatttgt tcaatgaaca cttactaaaa 44880 gcaacacaaa cagggtacaa gggatgcagt aacaaaagat acagggttca gaagagctct 44940 caggttatga ggatgatgga catgaaaaca ctccaattta gtacaactca atgttataat 45000 cctcacctga acgccctgct aagggagcct ggaggggagc tccctgagca ctcacactcc 45060 ttgggcattt acagttttca ctacccctcc caagttactt catggagtaa cttaagttgg 45120 ggacacctgt ggtctgggta ttgccctcca agccacttgg ccactcccac cccagttctc 45180 ccaatgcagt tccaagggta aggcctatga agccatctcc atctatatgg tggtggtctt 45240 ccctcatcct gatcttagtg ccctgtcata tcacaagata ggaggtagga gatacaggtg 45300 gtaacacttg tcaagctgat tccttggagg gaagaggtaa ggaagacagt gagaagttaa 45360 ccaccagctt tccttggctt cccccacccc caggtgaaag tgatgcgcag cctggaccac 45420 cccaatgtgc tcaagttcat tggtgtgctg tacaaggata agaagctgaa cctgctgaca 45480 gagtacattg aggggggcac actgaaggac tttctgcgca gtatggtgag cacaccaccc 45540 catagtctcc aggagccttg gtgggttgtc agacacctat gctatcacta ccctaggagc 45600 ttaaagggca gaggggccct gctttgcctc caaaggacca tgctgggtgg gactgagcat 45660 acatagggag gcttcactgg gagaccacat tgacccatgg ggcctggacc acgagtggga 45720 cagggctcaa cagcctctga aaatcattcc ccattctgca ggatccgttc ccctggcagc 45780 agaaggtcag gtttgccaaa ggaatcgcct ccggaatggt gagtcccacc aacaaacctg 45840 ccagcagggc gagagtaggg agaggtgtga gaattgtggg cttcactgga aggtagagac 45900 cccttcctat gcaacttgtg tgggctgggt cagcagctat tcattgagtt tgtctgtgtc 45960 actgaaactg accccagcca actgttctca gttcacagcc ctgttttcaa agaattacac 46020 atctctaaag gcaaacaggg cacggacaag gcaaactgga gaggcaaact gtagcctgag 46080 atggcctggg cttgccatca caggtattca ggtgctgagg gcccttagac caactagagc 46140 acctcactgc ctaggaaatc aatgaagggg aaatgagttc tagcggagcc ctgaaggatc 46200 agaattggat aaagttctta ttggcagaga ggcaccagga ttgaagtgac aggagcaaag 46260 acctgggagg aaagaggaga aaatcatcta tttcacctgg aaacaaatga ttccaagcat 46320 agaaataata acagctgaca agtactgagt gccctctata tgctaggcac tgggctgagg 46380 gattaacatg catgtgcatg tttattcctc atgacaacct tggtttccag ataagctgga 46440 ctggaaaggg acagagctgg gatcctgggc taatcagtct ggtcgccaag cctgagactt 46500 tagccactgc ccttcacatg ggggtccatg aaaatagtag tagtctggaa cagtttgggg 46560 gtacatcaag gtcgctgtgt tttaagctat ggagtctgga ctataggaga caaatgtaaa 46620 agagtttttt ggttgactgg ctttttggtt tttttgtttg tttgtttgtt tgtttgtttg 46680 tttgtttgtt ttttcctgtt tctggggctt gaatcaggaa ggaggttttt ttgttgttgt 46740 tgttttgaga aaggatattg ctctgttgcc cagactggag tgcagtggca cgatcatggc 46800 tcactacagc ttcgacctcc tgggctcaag caatcctcct gccttagcct cccaagtagc 46860 tggactacag gtgtgtacca ccacacctaa ttttttgaat ttttttttct tttttttttt 46920 tttttttttt ggtagagaca ggttctcact ttgttgccca ggcctgaatc tcaaactcct 46980 gggctcaagc attcctcctg cctcgccctc ccaaagtgtt gggattacag ttgtgagcca 47040 ccatgcccgg caggaaaaga tttttaagca agaaagctta agagctgtgg tttttccaaa 47100 atgagtctgg gctggcacag tggctcatgc ctgtaatccc agcacttttt tgggaggccg 47160 aggtgagtgg atcacttgag gtcaggagtt tgagaccagc ctggccaact ggtgaaaccc 47220 ctgtttctac taaagaaaaa aatgcaaaaa ttagctgggc gtggtggtgc acgcctgtag 47280 tcccagctac tcaggaggcc gaggcaggag aatagcttga acctgggagg cagaagttgc 47340 agtgagccaa gatcacacca ctgcattcca gcctgggtga cagagtgaga cttcatctca 47400 aaaaaaaaaa aaaagagaga ctgatatggt tagtacattg gggtggaatg cggagggtcc 47460 agggaatgga gccctgcata gggggctaat gaaacatttc agatttctga attaaggtag 47520 tggctgtggg gacaggagcc tgggaggcag ggtggagtca gaatggagag actggttggc 47580 aatgagggaa caggaggagg aggaggagga gttacgagtg gcttgaggtg tcacttacca 47640 gacatttggg ggatggggga tagccgtgat tgttgagcaa ctggtttggg aagagctagc 47700 attgatccct gctgttctgt gctagcagaa cctatcagca tcttctgggc aggaaactgg 47760 ctccatgaga ctggcttagg gagaggctgc tagtcaccta atctgcagag aaggggcagc 47820 tggagctgtg ggacagaaga ggcatccatg tagctggtgg gggtgtctca gcttgtgaag 47880 aggagatggc tttgagcagg gctgacactg aaaaggctgg aagaaaaaaa cagacacaca 47940 agagtctcag gatcaggtag cataggaaag ttgtggacag tctttgagga gcactccctc 48000 aggcaggcag gcaggcaggt catgagctat agcgattcag gaagagctcc ctgggtgtgt 48060 gagcagctcc aggagcctaa gggatgaaag tagtattgca gggggctgga gagcaaggag 48120 tggctccttc tacatttgca agggaaggag aaaggaagtt gctcctgaga gtggtaagag 48180 tcagtggtgg aggcctggag aggagacata acaaacaaat ttgttgacaa acattttggt 48240 aggaaggggg agagcttaaa gtttagacag tggggaaggt ggagtcttag aggaggtgaa 48300 tgtctgaaag acagagctag ctggagcaag aagtcacttc tctgttgcag gcaggaagga 48360 tccaaagtgg ctcaagccag agattgggag agtggggagg agggagcagc ctggatctaa 48420 gtaaaatggg tagaggtgga gggggtgctg caacggccag ggttttctga agttggggac 48480 attaggagag agctgtgagg gctttggcca gccactgtgc tagtgattgg tgaaccaaag 48540 gatgggcagg agatggcagc agggaagcag aggaagtcca ggcttcctgt tggtattggg 48600 acaagggaga ggccatagga ggccctggcc ctgttgtcca ggttgggttc tgaagctggg 48660 tgggcatggc ctggtaggag agcatctatg gcgcccaatt ccagattcag ggtctagttg 48720 atttgctggc cctgtagcct cagctcatgc ttctgttcca ggcctatttg cactctatgt 48780 gcatcatcca ccgggatctg aactcgcaca actgcctcat caagttggta tgtcccactg 48840 ctctgggcct ggcctccagg gtcctatcct tcctggcttc cttgtcacaa aggaggctga 48900 cttgtcccct ctggctagag ggcagaggtg ttgcctagga gctcctatct ttcccttcct 48960 gcttcttcca atgcccttct ctgtcctctg ggagctccga gacacacaca gacataattt 49020 caccttctct cattagcaac ctttgaaata atttgattag aagggacttc agaagtttgt 49080 tgactatatg tagaaaaccc tgtcatttta cctgcttttg ccccatagta gtcttgtaaa 49140 acagttcatt gctgacccca ttttacagtg gtggcacctg aagcctcagc ctgaggccac 49200 cgagctagta aatttacagg gaccagtttg agaccagcat tcctcccact gcccctcagc 49260 tgtggtggtt acaatgttgt ttgtcttact gacttgctat ctggcttcct gggtgtctac 49320 cggctggccc tggctctgcc ctctagaccc acaccacgca atcttcattc ctttcccaca 49380 tgactgccct gtagctattc aaagagcttg tctcccccaa gtctccccat ctactgcctc 49440 caccttgcct ttttctgtct tatcctggtt ctagccactg cctgaaatca ttttaggaat 49500 aagacaggac agggaaaaac aaaagcaacc ccctgtccca cctctgagtt ccactctcca 49560 agtccctgag cctcacctcc agggctccag tggctctgcc atgaacccac tgtgggctgg 49620 gagtctgctg tgcacagata ccagaccctc agaaacacaa atgccaagtg tgtctgtttt 49680 tttgttttgt tttgttttgt tttttagatg gagtctcatt ctgtttccca ggctggagtg 49740 cagtggtgca atcttggctt actgcagcct ctacctcccg ggttctagtg attgttctgc 49800 ttcagcctcc cagtagctag gactacaggc gtgtgccacc acgcccagct aatttttttt 49860 tttttttttt tgtattttta gtagagacag ggttttgcca tgttggccag gctggtcttg 49920 aactcctgac ctcaggtgat tcacccgcct tggcctccca aagttctggg attacaggtg 49980 gaagccaccg tgcctggcct gagtgtgtct atttgataga gctttctgct ctgattctcc 50040 cttgctatac accttttctc cccttctcag tggcttctct tgcctatgct tcctccccag 50100 ggccaggttt gagaacatcc ccatgaagtc ctgacctgtc ttttatccta ccaggacaag 50160 actgtggtgg tggcagactt tgggctgtca cggctcatag tggaagagag gaaaagggcc 50220 cccatggaga aggccaccac caagaaacgc accttgcgca agaacgaccg caagaagcgc 50280 tacacggtgg tgggaaaccc ctactggatg gcccctgaga tgctgaacgg tgagtcctga 50340 agccctggag gggacacccg cagagggagg acagatgctg cccttgcatc agagccctgg 50400 gaattccagg ggaggcctgt gaagcgtagg accggatacc cagagctgag gatatttttc 50460 ccttgccagg tggggcctca cgatttagct cctgagctca gggggctggg aactgatcag 50520 tgtcccatca tgggggataa ggtgagttct gactgtggca tttgtgcctc agggatcgct 50580 aagagctcag gctattgtcc cagctttagc cttctctctc catggtgaga actgaagtgt 50640 ggtgccctct ggtggataat gctcaaacca accagagatg ctggttggga ttcttgaaat 50700 cagggttgtg aggcctcaga aatggtctga atacaatcca ttttggagtc tgaggcccag 50760 agaagttcag tgaattgcct aggagcatac agctgcctaa tggcagaggc tagatgaacc 50820 ctagtctggt tcttttccac tttaacgtgc agtttcatcc taggcagtgt tatgttataa 50880 gggctctcca aggcagttca cctacggctg aggaaggact attttcaggt ggtgtctgcg 50940 caggacagcc tgtggggtgt ccctacagaa cctgttctag ccctagttct tagctgtggc 51000 ttagattgac cctagaccca gtgcagagca ggtaagggat gtaaacttaa cagtgtgctc 51060 tcctgtgttc cccaaggaaa gagctatgat gagacggtgg atatcttctc ctttgggatc 51120 gttctctgtg aggtgagctc tggcaccaag gccatgcccg aggcagcagg cctagcagct 51180 ctgccttccc tcggaactgg ggcatctcct cctagggatg actagcttga ctaaaatcaa 51240 catgggtgta gggttttatg gtttataacg catctgcaca tctttgccac gttcgtgttt 51300 cattggtctt aagagaagga ctggcagggt ttttttgttt tagatggagc ctcacttcgt 51360 tgcccaggct ggagtgcagt ggcacaatct gggctcactg caacctctgc cttctgggtt 51420 caagtgattc tcctgcctca gcctcccaag tagctgggac taccggcaca caccaccatg 51480 cccggctaat ttttgtattt ttagtagaga cagggtttca ccatgttggc caggctggtc 51540 ttgaactccg gacctcaggt gatccgcctg cctcagcctc taaaagtgct ggaattaata 51600 ggcgtgagct acctcgcccg gccaggtttt tttttttttt tttttagttg aggaaactga 51660 ggcttggaag agggcagtgg cttgcacatg gtcgataagg ggcagatgag actcagaatt 51720 ccagaaggaa gggcaagaga ctgttcatgt ggctgtctag ctagctcttg ggccaaatgt 51780 agcccttctc agttcccttc aagtagaagt agccactcta ggaagtgtca gccctgtgcc 51840 aggtaccacg tggacagagt gaggaatctt ggaaagattc ctacctttag gagtttagtc 51900 aggtgacagc atatctcagc gactcaaaca cacacacatt caaagccttc tgtaattcct 51960 acaaagttgt gaggggtaga ggagaggaga gacaagggat ggttaggata atgaaggaat 52020 gttttgtttt tgtttttgtt tttgagatgg agtttcactc tgtcacccag gctggagtgc 52080 agaggtgcaa tcttggctca ctgcagcctc cgcctcccag gttcaagcaa tcctcctgcc 52140 tcagcctccc aagtagctgg gactacaggt gtgcgccacc acgcctggct aatttttgta 52200 ttttcagtag agacagggtt tcgccatatt ggccaggctg gtctcaaatg cctgacctca 52260 ggtgatacac ccgcttcagc ctcccaaagt gctgagatta caggcatgag ctaccgtgcc 52320 tggccatgaa ggaagatttg ttttaaaaaa ttgttttctt taatattaat tgaacacctc 52380 tgttcagagc actgggctgg tgccagaggg tttcagacat gaatcagatc cagcacctca 52440 tagagcctta atctggcaca cacacacagc cacaaggaga cacagacaag gcagggtagg 52500 atgagtggaa gctaggagca gatgctgatt tggaacactt ggcttctgca gtgaagcccc 52560 ttcttagtcc tcttcagtaa cccagctctc agtggataca ggtctggatt agtaagattt 52620 ggagagatga ttggggattg gggagagctc tctaacctat tttaccacct cctcttctgc 52680 cattcttcct gtccacatcc ccagcatccc tttcccttgc caagtatctg tggcctctgt 52740 agtcctttgt aaacagctgt cttcttaccc tacagatcat tgggcaggtg tatgcagatc 52800 ctgactgcct tccccgaaca ctggactttg gcctcaacgt gaagcttttc tgggagaagt 52860 ttgttcccac agattgtccc ccggccttct tcccgctggc cgccatctgc tgcagactgg 52920 agcctgagag caggttggta tcctgccttt ttctcccagc tcacagggtc ctgggacgtt 52980 tgcctctgtc taaggccacc cctgagccct ctgcaagcac aggggtgaga gaagccttga 53040 ggtcaagaat gtggctgtca acccctgagc catctgacaa cacatatgta caggttggag 53100 aagagagagg taaagacata gcagcaagta atctggatag gacacagaaa cacagccatt 53160 aaaagaaagt ttaaaagaag gaaattcacc caaaccattt gaatacagta agtgtattca 53220 tctttcgata ttcccctgtc catatctaca catatacttt tttttatagt aaatagttct 53280 gtattttgcc ctgcatttcc cttgtgttta ctatccagtc ttcctgttta tcatttttgt 53340 cgacaacatg aaattctatt gagagactgt ctgaacatat tgtaatgtag atgttcaggt 53400 ttttccagtt tctctttaca ataggtattt aactacagtg agcagtttta tgcatttagc 53460 taatttctcc tttgaggaag tattttcaaa attaccttta ttcttctcag gtaataattt 53520 cattattacc aaagttaccc taggtctttt caagtgtgtg gttaaaaaac gagaatctgg 53580 ctgggcgcga tggctcacac ctgtaatccc agcactttgg gaggctgagg ctggtggatc 53640 acctgaggtc tggagttcga gaccagcctg gccaacatgg tgaaacccca tctctactaa 53700 aaatacaaaa cttagccagg catggtggca ggtgcctgta accccagcta cttgggaggc 53760 tgaggcagga gaattgcttg aacccagggg cggaggttgc agtgagccga tatcacgcca 53820 ttgcactcca gcctcggcaa caagagtgaa actctgtctc aaaaatgggg ttcttttcct 53880 gccatcaaaa atcatgtttc ttttaaaaac aagttcaaac attaccaaag tttatagcac 53940 aggaaatacg tcttctgtaa tctcccttaa ccaatatatc cctcaacatt ctcctcaccc 54000 ccaactccac cctcccagga taaccagttg ggacataatc tttatttaaa aatggtttcc 54060 ggatagagaa agcgcttcgg cggcggcagc cccggcggcg gccgcagggg acaaagggcg 54120 ggcggatcgg cggggagggg gcggggcgcg accaggccag gcccgggggc tccgcatgct 54180 gcagctgcct ctcgggcgcc cccgccgccg ccctcgccgc ggagccggcg agctaacctg 54240 agccagccgg cgggcgtcac ggaggcggcg gcacaaggag gggccccacg cgcgcacgtg 54300 gccccggagg ccgccgtggc ggacagcggc accgcggggg gcgcggcgtt ggcggccccg 54360 gccccggccc ccaggccagg cagtggcggc caaggaccac gcatctactt tcagagcccc 54420 ccccggggcc gcaggagagg gcccgggctg ggcggatgat gagggcccag tgaggcgcca 54480 agggaaggtc accatcaagt atgaccccaa ggagctacgg aagcacctca acctagagga 54540 gtggatcctg gagcagctca cgcgcctcta cgactgccag gaagaggaga tctcagaact 54600 agagattgac gtggatgagc tcctggacat ggagagtgac gatgcctggg cttccagggt 54660 caaggagctg ctggttgact gttacaaacc cacagaggcc ttcatctctg gcctgctgga 54720 caagatccgg gccatgcaga agctgagcac accccagaag aagtgagggt ccccgaccca 54780 ggcgaacggt ggctcccata ggacaatcgc taccccccga cctcgtagca acagcaatac 54840 cgggggaccc tgcggccagg cctggttcca tgagcagggc tcctcgtgcc cctggcccag 54900 gggtctcttc ccctgccccc tcagttttcc acttttggat ttttttattg ttattaaact 54960 gatgggactt tgtgttttta tattgactct gcggcacggg ccctttaata aagcgaggta 55020 gggtacgcct ttggtgcagc tcaaaaaaaa aaaaaaaaat gatttccagc ggtccacatt 55080 agagttgaaa ttttctggtg ggagaatcta taccttgttc ctttataggc caaggaccgc 55140 agtccttcag taacaccagt gtaaaagctt gaggagaaat tgtgaagcta cacagtattt 55200 gttttctaat acctcttgtc attctaaata tctttaattt attaaaaaat atatatatac 55260 agtattgaat gcctactgtg tgctaggtac agttctaaac acttgggtta cagcagcgaa 55320 caaaataaag gtgcttaccc tcatagaaca tagattctag catggtatct actgtatcat 55380 acagtagata caataagtaa actatattga atattagaat gtggcagatg ctatggaaaa 55440 agagtcaaga caagtaaaga cgattgttca gggtaccagt tgcaatttta aatatggtcg 55500 tcagagcagg cctcactgag gtgacatgac atttaagcat aaacatggag gaggaggagt 55560 aagcctgagc tgtcttaggc ttccggggca gccaagccat ttccgtggca ctaggagcct 55620 ggtgtttccg attccacctt tgataactgc attttctcta agatatggga gggaagtttt 55680 tctcctattg tttttaagta ttaactccag ctagtccagc cttgttatag tgttacctaa 55740 tctttatagc aaatatatga ggtaccggta acattatgcc catttctcac agaggcacta 55800 ctaggtgaag gagtttgcct gacgttatac aaccaggaag tagctgagcc tagatccctt 55860 ccacccaccc catggccctg ctcatgttcc acctgcctct aatttacctc ttttccttct 55920 agaccagcat tctcgaaatt ggaggactcc tttgaggccc tctccctgta cctgggggag 55980 ctgggcatcc cgctgcctgc agagctggag gagttggacc acactgtgag catgcagtac 56040 ggcctgaccc gggactcacc tccctagccc tggcccagcc ccctgcaggg gggtgttcta 56100 cagccagcat tgcccctctg tgccccattc ctgctgtgag cagggccgtc cgggcttcct 56160 gtggattggc ggaatgttta gaagcagaac aagccattcc tattacctcc ccaggaggca 56220 agtgggcgca gcaccaggga aatgtatctc cacaggttct ggggcctagt tactgtctgt 56280 aaatccaata cttgcctgaa agctgtgaag aagaaaaaaa cccctggcct ttgggccagg 56340 aggaatctgt tactcgaatc cacccaggaa ctccctggca gtggattgtg ggaggctctt 56400 gcttacacta atcagcgtga cctggacctg ctgggcagga tcccagggtg aacctgcctg 56460 tgaactctga agtcactagt ccagctgggt gcaggaggac ttcaagtgtg tggacgaaag 56520 aaagactgat ggctcaaagg gtgtgaaaaa gtcagtgatg ctcccccttt ctactccaga 56580 tcctgtcctt cctggagcaa ggttgaggga gtaggttttg aagagtccct taatatgtgg 56640 tggaacaggc caggagttag agaaagggct ggcttctgtt tacctgctca ctggctctag 56700 ccagcccagg gaccacatca atgtgagagg aagcctccac ctcatgtttt caaacttaat 56760 actggagact ggctgagaac ttacggacaa catcctttct gtctgaaaca aacagtcaca 56820 agcacaggaa gaggctgggg gactagaaag aggccctgcc ctctagaaag ctcagatctt 56880 ggcttctgtt actcatactc gggtgggctc cttagtcaga tgcctaaaac attttgccta 56940 aagctcgatg ggttctggag gacagtgtgg cttgtcacag gcctagagtc tgagggaggg 57000 gagtgggagt ctcagcaatc tcttggtctt ggcttcatgg caaccactgc tcacccttca 57060 acatgcctgg tttaggcagc agcttgggct gggaagaggt ggtggcagag tctcaaagct 57120 gagatgctga gagagatagc tccctgagct gggccatctg acttctacct cccatgtttg 57180 ctctcccaac tcattagctc ctgggcagca tcctcctgag ccacatgtgc aggtactgga 57240 aaacctccat cttggctccc agagctctag gaactcttca tcacaactag atttgcctct 57300 tctaagtgtc tatgagcttg caccatattt aataaattgg gaatgggttt ggggtattaa 57360 tgcaatgtgt ggtggttgta ttggagcagg gggaattgat aaaggagagt ggttgctgtt 57420 aatattatct tatctattgg gtggtatgtg aaatattgta catagacctg atgagttgtg 57480 ggaccagatg tcatctctgg tcagagttta cttgctatat agactgtact tatgtgtgaa 57540 gtttgcaagc ttgctttagg gctgagccct ggactcccag cagcagcaca gttcagcatt 57600 gtgtggctgg ttgtttcctg gctgtcccca gcaagtgtag gagtggtggg cctgaactgg 57660 gccattgatc agactaaata aattaagcag ttaacataac tggcaatatg gagagtgaaa 57720 acatgattgg ctcagggaca taaatgtaga gggtctgcta gccaccttct ggcctagccc 57780 acacaaactc cccatagcag agagttttca tgcacccaag tctaaaaccc tcaagcagac 57840 acccatctgc tctagagaat atgtacatcc cacctgaggc agccccttcc ttgcagcagg 57900 tgtgactgac tatgaccttt tcctggcctg gctctcacat gccagctgag tcattcctta 57960 ggagccctac cctttcatcc tctctatatg aatacttcca tagcctgggt atcctggctt 58020 gctttcctca gtgctgggtg ccacctttgc aatgggaaga aatgaatgca agtcacccca 58080 ccccttgtgt ttccttacaa gtgcttgaga ggagaagacc agtttcttct tgcttctgca 58140 tgtgggggat gtcgtagaag agtgaccatt gggaaggaca atgctatctg gttagtgggg 58200 ccttgggcac aatataaatc tgtaaaccca aaggtgtttt ctcccaggca ctctcaaagc 58260 ttgaagaatc caacttaagg acagaatatg gttcccgaaa aaaactgatg atctggagta 58320 cgcattgctg gcagaaccac agagcaatgg ctgggcatgg gcagaggtca tctgggtgtt 58380 cctgaggctg ataacctgtg gctgaaatcc cttgctaaaa gtccaggaga cactcctgtt 58440 ggtatctttt cttctggagt catagtagtc accttgcagg gaacttcctc agcccagggc 58500 tgctgcaggc agcccagtga cccttcctcc tctgcagtta ttcccccttt ggctgctgca 58560 gcaccacccc cgtcacccac cacccaaccc ctgccgcact ccagccttta acaagggctg 58620 tctagatatt cattttaact acctccacct tggaaacaat tgctgaaggg gagaggattt 58680 gcaatgacca accaccttgt tgggacgcct gcacacctgt ctttcctgct tcaacctgaa 58740 agattcctga tgatgataat ctggacacag aagccgggca cggtggctct agcctgtaat 58800 ctcagcactt tgggaggcct cagcaggtgg atcacctgag atcaagagtt tgagaacagc 58860 ctgaccaaca tggtgaaacc ccgtctctac taaaaataca aaaattagcc aggtgtggtg 58920 gcacatacct gtaatcccag ctactctgga ggctgaggca ggagaatcgc ttgaacccac 58980 aaggcagagg ttgcagtgag gcgagatcat gccattgcac tccagcctgt gcaacaagag 59040 ccaaactcca tctcaaaaaa aaaaa 59065 4 265 PRT Human 4 Leu Thr Glu Val Lys Val Met Arg Ser Leu Asp His Pro Asn Val Leu 1 5 10 15 Lys Phe Ile Gly Val Leu Tyr Lys Asp Lys Lys Leu Asn Leu Leu Thr 20 25 30 Glu Tyr Ile Glu Gly Gly Thr Leu Lys Asp Phe Leu Arg Ser Met Asp 35 40 45 Pro Phe Pro Trp Gln Gln Lys Val Arg Phe Ala Lys Gly Ile Ala Ser 50 55 60 Gly Met Ala Tyr Leu His Ser Met Cys Ile Ile His Arg Asp Leu Asn 65 70 75 80 Ser His Asn Cys Leu Ile Lys Leu Asp Lys Thr Val Val Val Ala Asp 85 90 95 Phe Gly Leu Ser Arg Leu Ile Val Glu Glu Arg Lys Arg Ala Pro Met 100 105 110 Glu Lys Ala Thr Thr Lys Lys Arg Thr Leu Arg Lys Asn Asp Arg Lys 115 120 125 Lys Arg Tyr Thr Val Val Gly Asn Pro Tyr Trp Met Ala Pro Glu Met 130 135 140 Leu Asn Gly Lys Ser Tyr Asp Glu Thr Val Asp Ile Phe Ser Phe Gly 145 150 155 160 Ile Val Leu Cys Glu Ile Ile Gly Gln Val Tyr Ala Asp Pro Asp Cys 165 170 175 Leu Pro Arg Thr Leu Asp Phe Gly Leu Asn Val Lys Leu Phe Trp Glu 180 185 190 Lys Phe Val Pro Thr Asp Cys Pro Pro Ala Phe Phe Pro Leu Ala Ala 195 200 205 Ile Cys Cys Arg Leu Glu Pro Glu Ser Arg Pro Ala Phe Ser Lys Leu 210 215 220 Glu Asp Ser Phe Glu Ala Leu Ser Leu Tyr Leu Gly Glu Leu Gly Ile 225 230 235 240 Pro Leu Pro Ala Glu Leu Glu Glu Leu Asp His Thr Val Ser Met Gln 245 250 255 Tyr Gly Leu Thr Arg Asp Ser Pro Pro 260 265 

That which is claimed is:
 1. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO:1; (c) a nucleic acid molecule consisting of the nucleic acid sequence of SEQ ID NO:3; and (d) a nucleotide sequence that is completely complementary to a nucleotide sequence of (a)-(c).
 2. A nucleic acid vector comprising a nucleic acid molecule of claim
 1. 3. A host cell containing the vector of claim
 2. 4. A process for producing a polypeptide comprising culturing the host cell of claim 3 under conditions sufficient for the production of said polypeptide, and recovering the peptide from the host cell culture.
 5. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:1.
 6. An isolated polynucleotide consisting of a nucleotide sequence set forth in SEQ ID NO:3.
 7. A vector according to claim 2, wherein said vector is selected from the group consisting of a plasmid, virus, and bacteriophage.
 8. A vector according to claim 2, wherein said isolated nucleic acid molecule is inserted into said vector in proper orientation and correct reading frame such that the protein of SEQ ID NO:2 may be expressed by a cell transformed with said vector.
 9. A vector according to claim 8, wherein said isolated nucleic acid molecule is operatively linked to a promoter sequence. 